Salmonella spp. in swine - the abattoir as a link in the food chain

Tese de doutoramento em Ciências Veterinárias 2012 Salmonella spp. in swine - The abattoir as a link in the food chain EDUARDA MARIA FREITAS GOMES DA SILVA NEVES EDUARDA MARIA FREITAS GOMES DA SILVA NEVES Salmonella spp. in swine - The abattoir as a link in the food chain Tese de Candidatura ao grau de Doutor em Ciências Veterinárias submetida ao Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto. Orientador – Doutor José Manuel Alves Correia da Costa Categoria – Investigador Principal com Habilitação Afiliação – Instituto Nacional de Saúde Doutor Ricardo Jorge Co-orientadora – Professora Doutora Maria de Fátima Moutinho Gärtner Categoria – Professora Catedrática Afiliação – Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto. Salmonella spp. in swine – The abattoir as a link in the food chain III Sê todo em cada coisa. Põe quanto és no mínimo que fazes. (in Odes de Ricardo Reis, Fernando Pessoa) Salmonella spp. in swine – The abattoir as a link in the food chain V Aos meus Pais Ao André, à Maria e à Francisca Salmonella spp. in swine – The abattoir as a link in the food chain VII DECLARATION/DECLARAÇÃO In accordance with the provision of the Portuguese law nº 230/2009 of 13 October, the candidate state that was involved in the study design, execution of experimental work, in the analysis and interpretation of results, and in their preparation for publication, presented in this work. No cumprimento do disposto no Decreto-lei nº 230/2009 de 13 de Outubro, como autora desta dissertação, declaro que participei na conceção dos estudos, execução experimental e interpretação dos resultados que estiveram na base do trabalho apresentado. The work presented in this thesis was performed in the National Laboratory of Veterinary Investigation (Laboratório Nacional de Investigação Veterinária – LNIV), in the Microbiology Laboratory of the Faculty of Pharmacy of the University of Porto and the National Reference Laboratory of Antimicrobial Resistances, Department of Infectious Diseases, National Institute of Health Dr. Ricardo Jorge. Articles already published and submitted and under revision or for publication in peer-reviewed scientific journals, were used in the elaboration of this dissertation. The presentation of each paper in this PhD dissertation does not necessarily reflect a chronological order, since some of the studies described below were done simultaneously. Publications and Manuscripts included in the thesis: SECTION III. RESULTS Chapter 1 Gomes-Neves E, Antunes P, Tavares A, Themudo P, Cardoso MF, Gärtner F, Correia da Costa JM, Peixe L (2012) Salmonella cross-contamination in swine abattoirs in Portugal: carcasses, meat and meat handlers. International Journal of Food Microbiology, 157, 82-87. Salmonella spp. in swine – The abattoir as a link in the food chain XIV Abstract Salmonella is one of the most important zoonotic agents worldwide, frequently implicated in foodborne diseases. Food of animal origin is identified as being the main vehicle for transmission to humans. Over the last years, it has been recognized that contaminated pork is a significant source of human infections. Thus, the aim of the present work was to characterize the occurrence of Salmonella in slaughter swine carcasses, meat and meat handlers, in Portuguese abattoirs, tracking cross-contamination and antimicrobial drug resistance in the isolates. Furthermore, we targeted the evaluation of meat handlers’ level of knowledge and practice, in order to clarify their participation throughout the cross-contamination process. The present study was developed in eight abattoirs and deboning rooms of districts of Braga and Porto, in the north of Portugal. One hundred slaughtered pigs were sampled, collecting ileoceacal lymph nodes, a carcass external surface swab and meat samples from each pig. The hands of the meat handlers responsible for deboning operations in the sampled carcasses were swabbed. A total of 345 samples was analyzed (300 pig samples and 45 hand samples). In Section III, Chapters 1 and 2 of results, 60 Salmonella isolates were phenotypically and genotypically characterized in order to access their antimicrobial resistance profiles and gain insight into the respective mechanisms of resistance; the genetic relatedness was established by pulsed-field gel electrophoresis (PFGE). The studies presented have shown a high frequency of Salmonella occurrence was found in the ileoceacal lymph node samples (26.0%), followed by carcass (16.0%) and meat samples (14.0%). However, ileoceacal lymph nodes that test positive for Salmonella are not found to be a predictor of positive test results further on in the process. Meat handlers were identified as a possible source of subsequent contamination, with 9.3% of the sample testing positive. Nine Salmonella enterica serotypes were detected, mainly S. Typhimurium (n=32) and the monophasic variant S. 4,[5],12:i:- (n=3), but also S. Derby (n=11), S. Rissen (n=4), S. Mbandaka (n=3), S. London (n=3), S. Give (n=2), S. Enteritidis (n=1) and S. Sandiego (n=1), belonged to 17 PFGE profile types corresponding to 12 clusters and 5 PFGE unique profiles. Antibiotic resistance was found in 75% of the clones, with 63% being multidrug-resistant (MDR). The highest resistance rates observed were to tetracycline (T, 70%), streptomycin (S, 63%), sulfamethoxazole (Sul, 62%), ampicillin (A, 57%) and chloramphenicol (C, 15%). The ASSuT (38%), ACSSuT (16%) and SSuT (13%) were the most frequent resistance phenotypes identified. S. 4,[5],12:i:- isolates, Salmonella spp. in swine – The abattoir as a link in the food chain XV which were recovered from swine in 3 abattoirs and clustered with S. Typhimurium isolates (PFGE cluster It), were mostly associated to ASSuT phenotype, related with blaTEM-1, strA- strB, sul2 and tet(B) resistance genes. However, one of the S. 4,[5],12:i:- isolates presented the resistance phenotype ST here, firstly, reported associated to strA-strB, tet(A) and tet(B) genes, which may be relevant in the future development of the increasing importance of the monophasic variant. S. Typhimurium DT104 (12%, PFGE cluster VIIt) isolated in lymph nodes, carcasses, meat and a meat handler in 1 abattoir, clearly demonstrating crosscontamination, was associated with the ACSSuT phenotype, and blaPSE-1, floR, aadA2, sul1 and tet(G) or tet(A) genes. S. Rissen isolates (PFGE cluster VIIIr), recovered in swine and meat handlers of 3 and 1 abattoir, respectively, differed in the MDR profile (T; ASuTW; ASSuTW; ACSSuTW), associated to tet(A), blaTEM-1, sul1 and/or sul3, aadA2, cmlA1 and dfrA12 genes. Otherwise, S. London, identified in 2 carcasses and a meat handler in 2 abattoirs is here, firstly, reported with the ANSSuT MDR profile, associated to blaTEM-1, strA- strB, sul2 and tet(A) genes. Integrons (37% of isolates were positive for class 1 integrons, 400-2000 bp), and resistance genes of the main human clones spreading worldwide, including Portugal, were identified in swine and abattoir environment, which might contribute to the load of MDR Salmonella and to the emergence of monophasic variant S.4, [5],12:i:-. Our results demonstrated that besides a high level of Salmonella swine contamination at the pre-harvest level, the slaughtering, cutting and deboning operations represents an important contribute to the occurrence of clinically relevant clones (e.g. S. Typhimurium DT104, the emergent S. 4,[5],12:i:- and S. Rissen) in pork products. This study also highlights the possibility of an ongoing MDR Salmonella community spread by abattoir workers. In the third chapter of results, (Section III, Chapter 3), a self-administered questionnaire, designed to assess “Knowledge” and “Practice” of public hygiene measures, was completed by meat handlers (MH) (n=159). Seventy-three per cent of the group had professional training in two different areas: Good Practice in Food Industry (GPFI, 12.03%), Work Safety and Hygiene (WSH, 22.8%), and both (37.9%). However, 24.5% have no professional training. The results of this study point to the need to improve training, particularly in Good Practice in Food Industry, since meat handlers with professional training in GPFI and in both areas had the highest proportions of correct answers. The development of evaluation criteria for the effectiveness of professional training is crucial to protect public health. Globally, the presented results reinforce the need of intervention strategies, preventing MDR Salmonella development in the pre-harvest stage, as well as the spreading in the food chain, through slaughter operations and meat handlers’ participation. Salmonella spp. in swine – The abattoir as a link in the food chain XVII Abbreviations attl – adjacent recombination site B cells – Bone marrow dependent lynfocytes CDC – Centers for Disease Control and Prevention cfu – colony forming units DNA – Deoxyribonucleic acid DT – Definitive phage type ECDC – European Centre for Disease Prevention and Control EFSA – European Food Safety Authority EU – European Union GALT – gut-associated lymphoid tissue GMP – Good manufacturing practice HACCP – Hazard Analysis Critical Control Points ICBAS – Instituto de Ciências Biomédicas Abel Salazar int – integrase gene IR – Inverted-repeated sequences IS – Insertion sequence ISCR – Insertion sequence common region MGE – Mobile genetic elements MS – Member states Pc – promoter PCR – Polymerase chain reaction PFGE – Pulsed-field gel electrophoresis PRRSV – Porcine reproductive and respiratory syndrome virus R type – Resistance type T cells – Thimus dependent lynfocytes u.v. – Ultraviolet USA – United States of America USDA – United States Department of Agriculture WHO – World Health Organization Salmonella spp. in swine – The abattoir as a link in the food chain XIX INDEx DECLARAÇÃO/DECLARATION .....................................................................................VII AGRADECIMENTOS / ACNOWLEDGMENTS ................................................................IX RESUMO ......................................................................................................................... XI ABSTRACT ................................................................................................................... XIV ABBREVIATIONS ......................................................................................................... XVII • SECTION I – INTRODUCTION / LITERATURE REVIEW ........................................... 1 1.Introduction ............................................................................................................... 3 2. Salmonella – A food-borne zoonosis of increasing importance ............................... 4 2.1. Historical aspects ............................................................................................ 4 2.2. Characteristics, taxonomy and nomenclature of Salmonella .......................... 5 2.3. Hosts and habitats .......................................................................................... 7 2.4.Transmission pathways .................................................................................... 9 3. Salmonella in Swine as a foodborne pathogen...................................................... 10 3.1. Infection and pathogenesis ........................................................................... 11 3.2. Vaccination and immunity ............................................................................. 14 3.2.1.Interactions of other microbial agents with Salmonella ....................... 15 3.3. Herd-level risk factors ................................................................................... 16 3.4. Current monitoring and control programs ..................................................... 16 3.5. The EU assessment: most frequent serotypes in humans, pigs and pork .... 17 3.5.1. Portuguese slaughter swine ............................................................... 19 4. Salmonella and antimicrobial resistance ................................................................ 21 4.1. Resistance phenotypes ................................................................................. 23 4.2. Resistance determinants and transmission .................................................. 24 Salmonella spp. in swine – The abattoir as a link in the food chain XX • SECTION II – Aims of the Thesis ............................................................................... 27 • SECTION III – RESULTS ........................................................................................... 31 Chapter 1. From Salmonella-carrier pigs to contaminated pork: from farm to fork. Salmonella cross-contamination in swine abattoirs in Portugal: carcasses, meat and meat handlers ..................................................................................................... 33 Chapter 2. One health: Antimicrobial Resistance entering the food chain. Clinically relevant multidrug resistant Salmonella enterica in swine, at slaughter: filling the gaps in the food chain ................................................................................. 41 Chapter 3. The weakest link: Meat handlers - from food producing animals to public health. Meat handlers training in Portugal: a survey on Knowledge and Practice ..... 51 • SECTION IV – GENERAL DISCUSSION .................................................................. 61 • SECTION V – CONCLUSIONS AND PERSPECTIVES ............................................ 73 • SECTION VI – REFERENCES .................................................................................. 77 Salmonella spp. in swine – The abattoir as a link in the food chain XXI List of Figures Figure 1. Comparison of the Salmonella serovar distribution in humans and animal sources in the EU. Salmonella EU baseline survey, 2008 (adapted from European Food Safety Authority, 2011c). Figure 2. Scatter diagram of the prevalence of Salmonella-positive breeding holdings versus the prevalence of Salmonella-positive producing holdings in the EU member states (from European Food Safety Authority, 2011c). Salmonella spp. in swine – The abattoir as a link in the food chain XXIII List of Tables Table 1. Examples of Salmonella serotypes and their host-specifity (from Österberg, 2010). Table 2. Observed prevalence of slaughter pigs infected with Salmonella in lymph nodes, in the EU and Norway, 2006-2007 (from European Food Safety Authority, 2008). Table 3. Frequency distribution of Salmonella serovars in lymph nodes samples of slaughter pigs in the EU and Norway, 2006-2007, data from Portugal (adapted from European Food Safety Authority, 2008). Table 4. Characteristics of the most important mobile genetic elements (MGEs) (from Manageiro, 2011). Salmonella spp. in swine – The abattoir as a link in the food chain 6 Velge, Cloeckaert, & Barrow, 2005). Although broad serotyping of all surface antigens can be used for formal identification, most clinical microbiological laboratories accomplish a few simple agglutination reactions to define specific O antigens into serogroups, designated groups A, B, C1, C2, D, and E (Farmer, 1995). The development of serotyping was fundamental for the understanding of the epidemiology of Salmonella infections (Mølbak et al., 2006). This grouping system can be used clinically to confirm genus identification; however, it cannot quickly identify whether the organism is likely to cause enteric fever, because considerable cross-reactivity among serogroups occurs (Chiu, Su, & Chu, 2004). The genus Salmonella is currently divided into two species: S. enterica and S. bongori, each of which contains multiple serotypes. S. enterica is further divided into six sub-species: enterica (I), salamae (II), arizonae (IIIa), diarizonae (IIIb), houtenae (IV) and indica (VI) (Popoff & Le Minor, 1997). The vast majority (99.5%) of strains of Salmonella isolated from humans and warm-blooded animals belong to sub-species I (Grimont & Weill, 2007), while the other five sub-species II-VI and S. bongori are primarily associated with cold-blooded animals and are only infrequently isolated from mammals (Foti et al., 2009; Nastasi, Mammina, & Salsa, 1999). S. enterica sub-species enterica contains all serovars able to promote illness in domestic animals and humans and only less than fifty serovars are responsible for most of the cases of disease (Denagamage, 2008). Members of this sub-species usually are named based on where the serovar or serotype was first isolated. For named serotypes, to emphasize that they are not separate species, the serotype name is not italicized, and the first letter is capitalized. At the first citation of a serotype, the genus name is given followed by the word “serotype” or the abbreviation “ser.” and then the serotype name (for example, Salmonella serotype or ser. Typhimurium). Subsequently, the name may be written with the genus followed directly by the serotype name (for example, Salmonella Typhimurium or S. Typhimurium) (Brenner, Villar, Angulo, Tauxe, & Swaminathan, 2000). Serotypes belonging to another sub-species are designated by their antigenic formulae, following the sub-species name (Popoff, Bockemuhl, & Gheesling, 2004). The simplified antigenic formulae of Salmonella serovars are listed in a document called the Kauffmann-White scheme (Popoff et al., 2003). The antigenic formulae of Salmonella serotypes are defined and maintained by the WHO Collaborating Centre for Reference and Research on Salmonella at the Pasteur Institute, Paris, France, and new serotypes are listed in annual updates of the Kauffmann-White scheme. Strains defined as S. Typhimurium possess two phases of H-antigens: in phase 1 this is H-antigen “I” and in phase 2 they are H-antigens “1, 2”. These are universally regarded as “classic” S. Typhimurium strains (antigenic formula: 1,4,[5],12:i:1,2). Antigenic variants that lack either the first or the second phase H antigen, or both, have been described (antigenic formulas respectively: 1,4,[5],12:-:1,2, or 1,4,[5],12:i:-, or 1,4,[5],12:-:-). Such variants have SECTION I – Introduction / Literature Review 7 been termed “Salmonella Typhimurium-like” strains. Within these Salmonella Typhimuriumlike strains, monophasic variants lacking the second phase H antigen (1,4,[5],12:i:-) are referred to as “monophasic S. Typhimurium” (European Food Safety Authority, 2010b). A second level of characterization is based on phage typing. By use of 37 different phages, serotype Typhimurium can be divided into more than 210 phage types (Anderson, Ward, Saxe, & De Sa, 1977; Botteldoorn, Herman, Rijpens, & Heyndrickx, 2004). Besides serotyping and phage typing, bacterial molecular typing methods, such as plasmid profiling, pulsed-field gel electrophoresis (PFGE), IS200 typing, ribotyping, random amplified polymorphic DNA analysis, and amplified fragment length polymorphism, are used for epidemiological investigation of salmonellae (Baggesen, Sandvang, & Aarestrup, 2000; Daly & Fanning, 2000; Ebner & Mathew, 2001; Liebana, Garcia-Migura, Breslin, Davies, & Woodward, 2001; Liebana et al., 2002; Olsen, Skov, Angen, Threlfall, & Bisgaard, 1997). These techniques are useful for describing clonal relationships between strains (On & Baggesen, 1997) and for assessing the distribution of Salmonella strains within food-processing environments (Botteldoorn et al., 2004; Giovannacci et al., 2001; Millemann, Lesage, Chaslus-Dancla, & Lafont, 1995). The use of polymerase chain reaction (PCR) assays for the identification of specific serotypes, as Salmonella enterica serotype Typhimurium DT 104 and U302 (Pritchett, Konkel, Gay, & Besser, 2000) and Salmonella enterica 4,[5],12:i:- (Soyer et al., 2009; Tennant et al., 2010) is also available. 2.3. Hosts and habitats Salmonellae can be frequently found in sewage, sea, and river water and can contaminate a variety of foods. The microorganisms have been isolated from many animal species including, cows, pigs, chickens, turkeys, pigeons, sheep, dogs, cats, horses, donkeys, seals, lizards and snakes (Foti et al., 2009; Grimont & Weill, 2007; Haraga, Ohlson, & Miller, 2008; Mandell, Douglas Jr, & Bennett, 1979; Mølbak et al., 2006). Furthermore, migratory birds, amphibians, fish and even insects can also be infected by Salmonella spp. (Foti et al., 2009; Greenberg, Kowalski, & Klowden, 1970; Mitscherlich & Marth, 1984; Wells, Boulton, Hall, & Bidol, 2004). Salmonella is generally regarded as part of the normal intestinal flora of reptiles kept as pets (Österberg, 2010; Warwick, Lambiris, Westwood, & Steedman, 2001), suggesting that wild terrestrial reptiles may work as reservoirs (Briones et al., 2004; Hidalgo-Vila, Díaz-Paniagua, de Frutos-Escobar, Jiménez- Martínez, & Pérez-Santigosa, 2007). Some Salmonella species are restricted to one or few animal species, whilst others have a wider host spectrum (Mastroeni et al., 2001). According to the European Food Safety Authority (EFSA), all serotypes of Salmonella enterica are potentially hazardous to human health and thus regarded as pathogens (European Food Safety Authority, 2010b). Nevertheless, the majority of Salmonella Salmonella spp. in swine – The abattoir as a link in the food chain 8 infections reported in humans, and domestic animals are caused by relatively few of the more than 2500 identified serotypes (Hendriksen et al., 2011; Österberg, 2010). Although most of the serotypes of Salmonella enterica sub-species enterica have the capability to colonize the alimentary tract of a wide range of animals, a few have a predilection for one or a few host species (Österberg, 2010). The serotypes may be divided into three groups: 1. host-specific serotypes, 2. host-restricted serotypes and 3. broad host range serotypes (Mastroeni & Maskell, 2006; Uzzau et al., 2000; Uzzau et al., 2001) (Table 1). The typhoid salmonellae (S. Typhi and S. Paratyphi A, B, and C) and S. Sendai remains important and exclusive pathogens in humans in developing countries and are able to cause a severe, systemic disease referred to as ‘enteric fever’ (Giaccone, Catellani, & Alberghini, 2012; Mølbak et al., 2006). Typhoid fever in humans is still endemic in many developing countries in Africa and Asia often owing to fecal contamination of water supply, affecting approximately 21 million individuals annually, with a mortality of 1% (Crump et al., 2003). Prevention of the disease by implementation of hygiene measures is possible, but can be difficult (Mastroeni et al., 2001). Table 1. Examples of Salmonella serotypes and their host-specifity (from Österberg, 2010) Group Serotype Main host Other host Host specific S Typhi, Human S Paratyphi Human S Abortusovis Sheep S Gallinarum Poultry S Abortusequi Horse Host restricted S Cholerasuis Swine Human S Dublin Cattle Human Broad host range S Typhimurium (ubiquitous) S Enteritidis The ability to survive outside the host is considered an essential piece of the epidemiology of Salmonella spp. In stored samples of feed, grass or dust, spiked with 106-108 colony-forming units (cfu) of S. Typhimurium per gram, survival times of one year are not uncommon and up to four years have been reported (Mitscherlich & Marth, 1984). In addition, in liquid manure, S. Typhimurium was re-isolated after 140 days at +10ºC (Gudding, 1975). In field experiments, the survival times have not been rather than long, but still at least weeks to months dependent on temperature and humidity (Holley & Guan, 2003; Semenov, Van Overbeek, & Van Bruggen, 2009). The feature of being able to survive and sometimes even replicate in varying environments promotes the ubiquitous SECTION I – Introduction / Literature Review 9 presence of Salmonella spp. and complicates its control. An element assumed to be essential for the persistence in the environment, as well as for the colonization in the host, is the biofilm formation defined as ‘bacterial communities enclosed in a self-producing matrix adherent to each other and/or surfaces or interfaces’ (Costerton, Lewandowski, Caldwell, Korber, & Lappin-Scott, 1995; Costerton, Stewart, & Greenberg, 1999). This multicellular structure allows the bacteria to adapt to divergent surfaces ranging from the epithelial cell layer in the intestine to the stainless steel in feed factories, meat plants or animal transport trucks. It is suggested that biofilm formation facilitate the persistence by protecting bacteria against environmental stress such as disinfection and desiccation (Vestby, Møretrø, Langsrud, Heir, & Nesse, 2009). These different habitats provide opportunities for adaptation and evolution, and this is revealed by the changing trends in salmonellosis observed in recent years (Newell et al., 2010). A further trend recently identified in Salmonella infections has been an increased association of outbreaks with previously unusual vehicles, like fresh produce. Many such harvests are produced in developing countries where manure is regularly used as a natural fertilizer. Recent studies suggest that some Salmonella spp. have now evolved to attach to and colonize vegetables (Barak, Gorski, Naraghi-Arani, & Charkowski, 2005; Franz & Van Bruggen, 2008; Islam et al., 2004; Klerks, Franz, van Gent-Pelzer, Zijlstra, & Van Bruggen, 2007). Thus, it seems that Salmonella spp. are remarkably adaptable organisms able to evolve to fill different niches and respond to environmental challenges, improving survival mechanisms and providing new host and novel habitat opportunities (Newell et al., 2010). 2.4. Transmission pathways Salmonellae may be transmitted through direct contact with infected animals or between humans, or from environments contaminated with feces. Transmission also occurs when organisms are introduced in food preparation areas and are allowed to multiply in food, due to inadequate storage temperatures, insufficient cooking, improper handling and cross contamination of ready-to-eat food (European Food Safety Authority, 2011c). Humans can be healthy carriers of S. enterica in the intestine. This may be a potential hazard to food hygiene, if the carriers are the people involved in producing and handling the food. Usually an asymptomatic carrier eliminates Salmonella in their feces for several months after the episode of gastroenteritis through which they became a carrier (Giaccone et al., 2012). Food-borne outbreaks of salmonellosis are consistently observed and regularly reported. This is a reflection of a low infectious dose in humans, an ability to grow in unprocessed food and in the environment allowing amplification, and long-term survival and, therefore, ease of recovery from contaminated foods (Giaccone et al., 2012; Newell et al., 2010). Salmonella spp. in swine – The abattoir as a link in the food chain 10 Although Salmonella spp. may survive for long periods in the environment, it is believed that the carrier animal is the major source of infection for both animals and humans (Fedorka-Cray, Gray, & Wray, 2000). The common reservoir of Salmonella is the intestinal tract of a wide range of domestic and wild animals, which results in a variety of foodstuffs, of both food of animal and plant origin, as sources of human infections. From the intestinal contents of livestock, the salmonellae can contaminate fresh meat, raw milk and egg shells (Giaccone et al., 2012). If the necessary hygienic precautions are not taken in the early stages of the production line (slaughter, milking, egg collecting), there is a risk that the salmonellae may then extent along the food chain, contaminating products such as cured meats, dairy and egg-based products, the main cause of salmonellosis in developed countries (Giaccone et al., 2012). Furthermore, through the feces of animals and man, salmonellae can contaminate farmland, surface water flow and vegetables if they are fertilized with animal manure or manure that is not properly fermented. Vegetables, therefore, can be a source of disease to humans just like fresh meat, milk, shell eggs and by-products (Franz & Van Bruggen, 2008; Klerks et al., 2007). Besides in animals, Salmonella can adhere well to the work surfaces, and from there extent to other foodstuffs by cross-contamination (Møretrø, Heir, Nesse, Vestby, & Langsrud, 2012). Additionally, contaminated animal feed may constitute a source of infection with Salmonella spp. in animals (Crump, Griffin, & Angulo, 2002; Davies, Scott Hurd, Funk, Fedorka-Cray, & Jones, 2004; Österberg, 2010). Salmonella in feed may derive from contaminated ingredients or from environmental contamination of the feed during crushing or subsequent feed production processes (Binter et al., 2011; O’Connor, Denagamage, Sargeant, Rajić, & McKean, 2008; Österberg, 2010). In recent EU data, it was detected from 0% to 3.6% in pig feed samples (European Food Safety Authority, 2012a). 3. Salmonella in swine and pork as a foodborne pathogen Food-producing animals, particularly poultry and swine, are considered to be the primary reservoir of non-typhoidal Salmonella, causing enteric infection in humans (Carattoli, 2008). Swine are frequently asymptomatic Salmonella carriers who play a main role as a primary source of contamination of the environment, other animals and fresh or processed meat (Gopinath, Carden, & Monack, 2012). Pork is an important source of Salmonella infections for humans, mostly related with S. Typhimurium (Boyen et al., 2008; Buchholz et al., 2005; Gebreyes, 2008; Gebreyes, Thakur, Davies, Funk, & Altier, 2004; Mølbak et al., 1999; Thakur, Tadesse, Morrow, & Gebreyes, 2007; Threlfall, 2000) and pork-related outbreaks with a fatal outcome have been described (Jansen, Frank, & Stark, 2007). SECTION I – Introduction / Literature Review 11 Indeed, in USA, statistical models have predicted that every year nearly 100,000 human cases of salmonellosis are related to the consumption of pork, with a resultant annual social cost of approximately 80 million dollar (Miller, Liu, McNamara, & Barber, 2005). In the EU, it is estimated that around 28% of the human salmonellosis cases are attributable to pigs and pork (European Food Safety Authority, 2011b). The Salmonella situation at the farm-level has recently started to become an issue in various countries, coinciding with growing concern regarding food safety and problems associated to large-scale industrial pork production (Crump et al., 2002; Davies, 1997; Davies et al., 2004; Fraser, 2006; Kich et al., 2007; Kich et al., 2011; Molla et al., 2010). Stress factors, such as feed withdrawal from swine prior to slaughter and transport to the abattoir, have been shown to promote shedding of Salmonella by carrier swine (Berends, Urlings, Snijders, & Van Knapen, 1996; Berends, Van Knapen, Mossel, Burt, & Snijders, 1998a, 1998b; Botteldoorn et al., 2004; Boyen et al., 2008; De Busser et al., 2011; Delhalle, Saegerman, Farnir, et al., 2009; Lo Fo Wong, Hald, van der Wolf, & Swanenburg, 2002; Lo Fo Wong et al., 2003; O’Connor et al., 2008; Swanenburg, Urlings, Snijders, Keuzenkamp, & Van Knapen, 2001; Swanenburg, Van der Wolf, Urlings, Snijders, & Van Knapen, 2001). This release of Salmonella contributes to the contamination of carcasses and the environment at the slaughterhouse, threatening meat consumers (De Busser et al., 2011; Delhalle, Saegerman, Farnir, et al., 2009; Delhalle, Saegerman, Messens, et al., 2009; Hald, Lo Fo Wong, & Aarestrup, 2007; Letellier, Messier, Paré, Ménard, & Quessy, 1999; Lo Fo Wong et al., 2003; Vieira-Pinto, Temudo, & Martins, 2005; Vieira-Pinto, Tenreiro, & Martins, 2006). The public health risk of Salmonella infection from ingestion of contaminated pork relies on multiple factors. Including the level of infection in the pig herd (European Food Safety Authority, 2011c; Hill et al., 2003; Nollet et al., 2005), hygiene during carcass processing in the abattoir (Berends et al., 1998a; Borch, Nesbakken, & Christensen, 1996; De Busser et al., 2011; Delhalle, Saegerman, Messens, et al., 2009; Swanenburg, Urlings, et al., 2001; Swanenburg, Van der Wolf, et al., 2001), meat storage and distribution conditions (Delhalle, Saegerman, Farnir, et al., 2009; Mann, Smith, & Brashears, 2004) and finally, the handling of undercooked pork by the consumer (Hill et al., 2003). 3.1. Infection and pathogenesis Swine usually get infected by oral route and can carry Salmonella asymptomatically in the tonsils, the intestines and the gut-associated lymphoid tissue (GALT) (Boyen et al., 2008; Fedorka-Cray et al., 2000; Mastroeni & Maskell, 2006; Scherer et al., 2008; Wood, Pospischil, & Rose, 1989). The transmission of the infection is facilitated by low hygiene standards and/or dense populations facilitating faecal contamination of feed or Salmonella spp. in swine – The abattoir as a link in the food chain 12 the environment (Österberg, 2010). Except for infections with Salmonella Typhisuis, Cholerasuis and some types of S. Typhimurium, Salmonella infections of pigs are practically always subclinical (Boyen et al., 2008; Huang, Lin, & Wu, 2009). Infections with the host-adapted serotype Choleraesuis often occur in North America and Asia being only occasionally described in Western Europe or Australia (Chang et al., 2005; Chiu et al., 2004; Fedorka-Cray et al., 2000; Nollet et al., 2006). Disease associated with this serotype is characterized by septicemia, enterocolitis or bacteremia localization as pneumonia and hepatitis or sporadically as meningitis, encephalitis and abortion (Boyen et al., 2008; Haesebrouck et al., 2004; Huang et al., 2009). In Western Europe, S. Typhimurium is responsible for most of the cases of clinical salmonellosis in swine (Boyen et al., 2008; Haesebrouck et al., 2004). This serotype is mainly related with enterocolitis including a febrile phase with dullness and loss of appetite, watery diarrhea and reduced general condition, followed by recovery with continued excretion of the bacteria for varying time periods (Boyen et al., 2008; Griffith, 2006). Although these infections may result in enteric and fatal systemic disease, this serotype frequently passes sub clinically in swine (Boyen et al., 2008; Haesebrouck et al., 2004). After infection with S. Typhimurium, pigs may develop a carrier state, excreting intermittently the bacteria for up to 28 weeks, in spite of a declining after the first 2 weeks, without presenting clinical signs (Haesebrouck et al., 2004; Scherer et al., 2008; Wood et al., 1989). Single oral doses of among 101- 103 cfu Salmonella spp. can be sufficient to infect about 0.1 to 10% of exposed animals. Additionally, as little as 2 cfu g-1 Salmonella spp. of feed may be sufficient to infect farm animals (Wray, Todd, McLaren, Beedell, & Rowe, 1990). After oral or aerogenic uptake of Salmonella bacteria, the tonsils and distal intestinal tract (ileum, caecum and colon) are colonized (Boyen et al., 2008; Fedorka-Cray, Kelley, Stabel, Gray, & Laufer, 1995; Marg, Scholz, Arnold, Rösler, & Hensel, 2001; Wood et al., 1989). The palatine tonsils are frequently severely infected and should, therefore, not be underestimated as a source of Salmonella contamination during slaughter (Kühnel & Blaha, 2004; Vieira-Pinto et al., 2005; Wood et al., 1989). The infection occurs in common in three different steps, after oral inoculation: (a) colonization of the gut and adhesion to the wall, (b) invasion of the wall, (c) dissemination to mesenteric lymph nodes and other organs (Berends et al., 1996; Boyen et al., 2008; Scherer et al., 2008). Underneath definite conditions, such as during transport, infections may also occur directly via the tonsils, whereby the agent, within 2- 6 h, can reach the colon and rectum via lymphatic routes (Berends et al., 1996; Reed, Olander, & Thacker, 1986). Colonization of the gut happens when enough numbers are ingested to pass through the stomach, or after multiplying in the oropharynx and tonsils (Wood et al., 1989). The acid environment of the stomach establishes an obstacle and diminishes the number of viable Salmonella bacteria (Giannella, Broitman, & Zamcheck, SECTION I – Introduction / Literature Review 13 1972; Haesebrouck et al., 2004). Every situation upsetting the pH of the stomach thus improves the number of salmonellae that reach the small intestine and stimulate effective colonization. In the proximal part of the small intestine, bile inhibits invasion of the mucosa by suppressing the Salmonella intestinal invasion mechanism (Galán, 2001; Haesebrouck et al., 2004; Prouty & Gunn, 2000), this might explain why Salmonella preferentially colonizes the ileum, caecum and colon (Boyen et al., 2008). In the intestinal wall, salmonellae are located in and between the enterocytes and in macrophages and leucocytes, but because bacteria can survive and proliferate in macrophages and leucocytes, translocation to lymph nodes and other organs will certainly happen (Wells, Maddaus, & Simmons, 1988). Neutrophils are attracted to the intestinal lamina propria and migrate towards the lumen. This process is related with the development of diarrhea (Boyen et al., 2008). The neutrophils in the gut belong to the first line of defense against a Salmonella infection, hence inefficient uptake by them may provide an opportunity for the pathogen to colonize and/or replicate to levels that enable development of a carrier state or clinical infection in pigs (Stabel, Fedorka-Cray, & Gray, 2002). The presence of high numbers of neutrophils in the porcine gut allows the host to overcome a Salmonella infection (Foster et al., 2003; Foster, Hulme, Lovell, Reed, & Barrow, 2005). Conversely, the damage induced by activated neutrophils is considered the main cause of the gut pathology distinctive for Salmonella infections (Boyen et al., 2008; Tükel et al., 2006). When the Salmonella bacteria have reached the intestinal lamina propria again they stimulate their uptake, this time by macrophages. Inside these macrophages, Salmonella is capable of surviving and even multiplying (Haesebrouck et al., 2004; Hensel, 2000). The safe position, which the macrophage offers, permits the bacteria to extent intra-cellularly all through the body and reaches the internal organs (Haesebrouck et al., 2004). Eventually, Salmonella induces an apoptosis-like process in the host macrophage (Van der Velden, Lindgren, Worley, & Heffron, 2000). This process permits the uptake of bacteria by other macrophages and consequently, stimulates bacterial dispersion (Haesebrouck et al., 2004). The macrophage death promotes fast dispersal in macrophages of the intestinal mucosa and spreading in the internal organs (Boyen et al., 2008). In systemic infections, the bacteria reach the phagocytes of the spleen, liver and bone marrow (Mastroeni et al., 2001), conversely there is no detection at these organs in Salmonella-carrier pigs (Scherer et al., 2008). Complement activation at the bacterial surface or the presence of opsonizing serum antibodies facilitates the uptake of the organisms by phagocytes (Liang-Takasaki, Saxen, Makela, & Leive, 1983; Mastroeni et al., 2001; Mastroeni & Maskell, 2006; Saxén, Reima, & Mäkelä, 1987). During systemic infections, the majority of salmonellae are associated with macrophages and polymorphonuclear phagocytes and the ability to grow within these cells seems to be a requirement for Salmonella virulence (Mastroeni et al., Salmonella spp. in swine – The abattoir as a link in the food chain 14 2001). When high bacterial numbers are extended in the tissues, salmonellae can be seen also in the extracellular compartment and in non-phagocytic cells, namely hepatocytes. (Conlan & North, 1992; Hsu, 1989; Mastroeni et al., 2001). Studies showed that pigs which were orally administered 109 cfu of several types of Salmonella spp. excreted the organisms within 24 h. In slaughter swine, at the moment the first animals were slaughtered (8 h after oral inoculation) their mesenteric lymph nodes were already positive (Berends et al., 1996; Wood et al., 1989). Furthermore, experiments with weaned pigs demonstrated that lymphoid tissues closely associated with the digestive tract, such as the tonsils and the mesenteric lymph nodes, may harbor Salmonella spp. for 28 weeks or longer, but that other lymph nodes, such as the axillary or inguinal lymph nodes, only contain them for a period of 2-4 weeks (Berends et al., 1996; Wood et al., 1989). Regarding the spread of infections between pigs, excretion in the feces is particularly important (Berends et al., 1996; Boyen et al., 2008; Scherer et al., 2008). 3.2. Vaccination and immunity In swine, the role of the immune-status of individuals concerning Salmonella is not completely clear. Host resistance to Salmonella relies initially on the production of inflammatory cytokines leading to the infiltration of activated inflammatory cells in the tissues (Mastroeni et al., 2001; Mastroeni & Ménager, 2003; Mastroeni & Maskell, 2006). Thereafter, T- and B-cell dependent specific immunity develops allowing the clearance of Salmonella microorganisms from the tissues and the establishment of long-lasting acquired immunity to re-infection. The increased resistance that develops after primary infection/vaccination requires T-cells cytokines in addition to opsonizing antibody (Mastroeni et al., 2001). Nevertheless, for reasons that are not completely understood, seroconversion and/or the presence of detectable T-cell memory does not always correlate with the development of acquired resistance to infection (Mastroeni et al., 2001). The best vaccine against S. Typhimurium prevents: (1) colonization; (2) shedding of the bacteria in the environment; (3) the development of carriers; and (4) clinical salmonellosis and promotes elimination of Salmonella bacteria from the infected porcine host (Haesebrouck et al., 2004). However, at this moment, as in poultry, vaccination against Salmonella in pigs seems only to reduce the infection pressure and is effective, especially in addition to other preventive measures taken at farm level and at the abattoir. In a more realistic approach, a vaccine should be able to: (1) prevent clinical symptoms, (2) reduce shedding by infected pigs and hence spreading to other pigs and (3) increase the threshold for infection of susceptible pigs with S. Typhimurium. An efficient vaccine should therefore contribute to the break of the infection chain (Haesebrouck et al., 2004). Wholecell killed vaccines and subunit vaccines are used in the prevention of Salmonella infection SECTION I – Introduction / Literature Review 15 in animals and in humans with variable results (Mastroeni & Ménager, 2003; Mastroeni & Maskell, 2006). Live Salmonella vaccines resultant of chemical or u.v. mutagenesis demonstrated to be immunogenic and protective, and are still in use, in spite of the need for repetitive parenteral administration (Mastroeni et al., 2001). Progress in the knowledge of the genetics of Salmonella virulence and modern recombinant DNA technology offers the opportunity to introduce multiple defined attenuating and irreversible mutations into the bacterial genome. This has lately allowed the development of Salmonella strains devoid of significant side effects but still capable of inducing solid immunity after single oral administration. Live attenuated Salmonella vaccines have been used for the expression of heterologous antigens/proteins that can be successfully delivered to the immune system (Mastroeni et al., 2001; Mastroeni & Ménager, 2003). Hence, live vaccine strains offer an improved protection against Salmonella infections compared to inactivated vaccines, possibly due to the more marked cellular immune response and the induction of mucosal IgA production (Boyen et al., 2008; Denagamage, O’Connor, Sargeant, Rajic, & McKean, 2007; Haesebrouck et al., 2004; Mastroeni et al., 2001). The evidence available suggests that Salmonella vaccines are related with reduced prevalence in swine at or near harvest. For instance, vaccination with a live modified S. Choleraesuis vaccine twice at 3 and 16 weeks of age was effective in reducing Salmonella prevalence in ileocecal lymph nodes at slaughter from 7.2% (unvaccinated barns) to 0.6% (vaccinated barns) (Denagamage et al., 2007; Maes et al., 2001). Moreover, effective vaccine-induced immunity requires control bacterial growth at each focus of infection and must hinder the redistribution of Salmonella to new foci (Mastroeni & Ménager, 2003). Knowledge of the anatomical sites where protective immunity must operate seems to be relevant to vaccine design (Mastroeni & Ménager, 2003). However, conclusions seem to be based on studies with design and reporting deficiencies that could potentially indicate biases with the outcome (Denagamage et al., 2007). In spite of the great recent advances in the development of Salmonella vaccines, a large proportion of the work has been conducted in laboratory rodents, and more research in other animal species seems to be required to improve effectiveness (Denagamage et al., 2007; Mastroeni et al., 2001). 3.2.1. Interactions of other microbial agents with Salmonella Bacterial (Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae) and viral (hog cholera virus, porcine reproductive and respiratory syndrome virus (PRRSV), Aujeszky’s disease virus) infections can result in immunodeficiency in pigs (Segalés et al., 2004). Additionally, some of these and also other swine pathogens (porcine parvovirus, swine influenza virus, African swine fever virus) are capable of replicate in different immune cells and damage their function. These infections may facilitate colonization by Salmonella, increased shedding or even higher mortality rates in pigs (Boyen et al., 2008). Salmonella spp. in swine – The abattoir as a link in the food chain 22 likely are resistant populations to develop among pathogens and commensal bacteria of an increasing number of animals in an exposed population (Jansen, Van der Bruggen, Verhoef, & Fluit, 2006; Johnsen et al., 2009; van Hoek et al., 2011). However, there is great diversity: whereas some bacteria very rapidly develop resistance, others remain susceptible (Gould, 1999; Phillips et al., 2004). Over the past six decades, bacterial populations have replied to the selective pressure of antimicrobial drugs by developing resistance to all commercially existing agents (Johnsen et al., 2009; Levin, 2001). The population dynamics of antimicrobial resistance depend upon the substances administered; resistance is also influenced by a number of other factors, not least: the availability of preexisting resistance genes, the exchangeability of the resistance genes and their functional activity in different bacterial hosts, and the selective pressure (Schwarz, Kehrenberg, & Walsh, 2001). Obviously, if antibiotics are present within the environment, there is strong selective pressure for the spread of resistance and those factors that promote the spread and gene transfer, for instance, is also more likely in environments where bacteria are in close proximity to each other and in relatively high density, such as the gut and oral cavity (van Hoek et al., 2011). Within Salmonella several multidrug-resistant (MDR) strains seem to have gained relative advantages as they have managed to spread rapidly in some animal and human populations, for example S. Newport in the USA, S. Typhimurium DT104 in Europe, and the monophasic variant 4,5,12:i:- of S. Typhimurium which is currently increasing in Europe, associated with pigs and pork (Butaye et al., 2006; European Food Safety Authority, 2012a, 2012b; Hauser et al., 2010; Hopkins et al., 2010). Within Salmonella, especially Salmonella enterica serotype Typhimurium, these MDR strains cause particular concern because of their increasing prevalence in humans becoming a worldwide health problem (Butaye et al., 2006; Parry & Threlfall, 2008; Shahada, Amamoto, Chuma, Shirai, & Okamoto, 2007; Shahada, Sugiyama, Chuma, Sueyoshi, & Okamoto, 2010). The antimicrobial agents’ widespread use in pigs during rearing created a selective pressure that may have contributed to the occurrence and dissemination of these MDR strains, which can be transmitted to humans through food products, particularly those of animal origin (Botteldoorn et al., 2004; Carattoli, 2008; Daly & Fanning, 2000; Threlfall, 2002). The campaign against what has been considered excessive clinical use has been generally directed at human and veterinary medicine. However, there has been also an intensive opposition on the food producing animals’ use of antibiotics, based upon the assumption that is imprudent and may act as an important source of resistance in bacteria affecting humans (European Commission, 1998, 1999; Levy, 1984, 2001; Threlfall, 2002; Witte, 1998; World Health Organization, 2001). In Europe, since the first harmonization by Directive 70/524 until Regulation (EC) No 1831/2003 on additives for use in animal SECTION I – Introduction / Literature Review 23 nutrition, there was, since 2006, a total banning of antibiotic growth promoters as a precaution (Castanon, 2007). The decreasing of the antibiotics used for animal production, and consequently, the reduction of the risk of transferring to human of bacterium with antibiotic-resistant genes, is the main expected result from this banning (Castanon, 2007). Otherwise, data suggested that the growth-promoter ban had determined an increase in infections and, consequently, a considerable growing through the use of therapeutic antibiotics for food animals in Europe, nevertheless, a reduced overall antimicrobial use in animals (McEwen, 2009). In Sweden, it was reported that as a result of the previous national banning in 1986 and an effort on disease prevention and correct use of antimicrobials, the total use of antibacterial drugs to animals diminished by approximately 55% in the period 1986-1999, and a comparatively low prevalence of antimicrobial resistance has been preserved (Wierup, 2001a). Recommended voluntary guidelines include orientation for prudent use among veterinarians, as well as information to farmers on cautious use for antibiotics that are sold over the counter (Höjgård & Vågsholm, 2010). For example, in Sweden, it is considered a good veterinary practice that drugs should only be prescribed to individual animals after a clinical and laboratory examination identifying the disease-causing agent. Another recommendation is that narrow spectrum antibiotics should be preferred to broad-spectrum drugs (Höjgård & Vågsholm, 2010). However, in antimicrobial free swine production systems, MDR S. Typhimurium strains were also detected, regardless of the absence of antimicrobial selection pressure (Thakur et al., 2007). 4.1. Resistance phenotypes Surveillance data demonstrated an increase in overall antimicrobial resistance among salmonellae from 20%-30% in the early 1990s to as high as 70% in some countries after the turn of the century (Boyen et al., 2008). The resistance rate, however, varies with distinct serotypes and different antibiotics, S. Enteritidis, one of the most prevalent Salmonella serotypes, is relatively more susceptible to antimicrobial agents than are other serotypes (Boyen et al., 2008). A much higher rate of resistance was found in S. Typhimurium, another globally prevalent serotype (Su et al., 2004). In the early 1990s, a distinct MDR strain of phage type 104 (DT104) S. Typhimurium strain was isolated and found to be simultaneously resistant to ampicillin, chloramphenicol, streptomycin, sulfonamide, and tetracycline, which corresponds to an ACSSuT phenotype (Boyen et al., 2008; Helms, Ethelberg, & Mølbak, 2005; Mølbak et al., 1999; Threlfall, 2002). Phage type DT104 is frequently isolated from pigs or pork (Gebreyes et al., 2004; Threlfall, 2000). Salmonella spp. in swine – The abattoir as a link in the food chain 24 In a recent EU data registered for Salmonella isolates from pigs (European Food Safety Authority, 2012b), resistance levels were 57% for tetracyclines, 55% for ampicillin and 59% for sulfonamides. Ciprofloxacin and nalidixic acid resistance levels were low, at 3% and 2% respectively, whereas the level of resistance to cefotaxime about 0.8%. Regarding pig meat, the resistance to tetracyclines (50%), ampicillin (47%) and sulfonamides (52%) was also common in Salmonella spp. isolates. Resistance to ciprofloxacin and nalidixic acid was 5% and 4%, respectively, and cefotaxime resistance equaled 0.2% (European Food Safety Authority, 2012b). Over the years 2005-2010, a relatively stable situation in resistance has been observed in Salmonella spp. isolates from pigs in the EU (European Food Safety Authority, 2012b). Regarding salmonellosis in EU, resistance in human Salmonella isolates, especially S. Typhimurium, was very high for ampicillin (64%), tetracyclines (58.5%) and sulfonamides (57.2%) and high for streptomycin (44.1%). Resistance to these antimicrobials in isolates from monophasic S. Typhimurium was extremely high, in all cases above 80% (European Food Safety Authority, 2012b). Due to the spread of resistance to conventional antibiotics, the currently recommended drugs of choice for treatment of salmonellosis in humans are fluoroquinolones and third-generation or extended-spectrum cephalosporins, but therapy may be also complicated by the fact that antimicrobial resistance in Salmonella isolates from these infections has become increasingly common (Chang et al., 2005; Chiu et al., 2004; Stoycheva & Murdjeva, 2006; Su et al., 2004). According to EU data, resistance to these clinically important antimicrobials was still relatively low, namely ciprofloxacin (8.6%) and cefotaxime (1.0%). Noteworthy, resistance to quinolones (ciprofloxacin and nalidixic acid) was higher in S. Enteritidis (9.3%, 18.7%) isolates than in S. Typhimurium isolates (4.7%, 8.9%) (European Food Safety Authority, 2012b). Recently, a new clonal group with resistances to ampicillin, streptomycin, sulfonamides, and tetracycline (ASSuT) was identified in Italy, United Kingdom and Denmark among human and animal strains of S. Typhimurium and its monophasic variant, suggesting its circulation in different European countries (Graziani et al., 2008; Lucarelli et al., 2010; Lucarelli et al., 2012). 4.2. Resistance determinants and transmission Antibiotic resistance is the best-known example of rapid adaptation of bacteria to a new ecosystem (Carattoli, 2001). The capability of bacteria to develop their ecological niche, also in the presence of certain antibiotics, can be elucidated by the acquisition of resistance genes by horizontal gene transfer and/or by the accumulation of point mutations leading to the alteration of existing genes (Carattoli, 2001; van Hoek et al., SECTION I – Introduction / Literature Review 25 2011). Several studies on bacterial pathogens of human and animal origin concluded that MDR is a consequence of horizontal gene transfer, mediated by bacteria transformation, transduction and conjugation (Carattoli, 2001). Nowadays, a worrying trend is the presence of genetic elements that co-integrate antibiotic resistance and virulence determinants, compromising the therapeutic options in cases of invasive Salmonella infections (Fluit, 2005). Indeed, a variety of transposable elements has been recently identified contributing to the dissemination of relevant antimicrobial resistance genes in Salmonella (Lucarelli et al., 2012; Miriagou, Carattoli, & Fanning, 2006; Stellwagen & Craig, 1998). It is well established that the distribution of antimicrobial resistance is often mediated by mobile genetic elements (MGEs), such as plasmids and/or transposons and/or insertion sequences (Lucarelli et al., 2012) (Table 4). Indeed, several of the antibiotic resistance genes observed in Gram negative Table 4. Characteristics of the most important MGEs (from Manageiro, 2011) Gene Transfer Element Characteristics of DNA transfer elements Plasmid Plasmids are transferable genetic elements capable of autonomous replication within a suitable host. Plasmids can be either self-transmissible (conjugative) or mobilisable (non-self-transmissible). Whereas the first group encodes a complete conjugative DNA transfer apparatus (Trafunctions), the second group usually bears only the functions required for initiation of its own transfer DNA rreplication (Mob functions). Insertion sequences Insertion sequences (IS) are the simplest transposable elements; by definition, IS carry only the genetic information necessary for insertion functions and no accessory genes (for example, drug resistance). IS elements are small genetic elements that are flnked by short terminal inverted-repeat sequences (IR) of 10-40 bp and are able to insert a multiple sites in target DNA ISCR Insertion Sequence Common Region (ISCR) elements are IS that have similarities to the IS91 family in both structure and function. These elements are known to move by a process called rolling-circle replication, and a function of this process is the concomitant movement of additional sequences found upstream of their transposase genes. Transposon Transposons are genetic elements that physically transpose from one genetic position to another, within the chromosome or plasmid in which they reside. Some transposons carry one or more antibiotic resistance genes in their central regions. Complex transposons contain IS with short IR at their termini; undergo replicative transposition Conjugative transposons, also called integrated conjugative elements, are integrated DNA elements that exercise thamselves to form covalentely ciosed circular intermediate. This circular intermediate can either reintegrate in the same cell or transfer by conjugation to a recipient and integrate into the recipient's genome. Integron Integrons are DNA elements, not self transmissible, with the ability to capture genes, by site-specific recombination. Integrons have an integrase gene (inf) to mediate excision and orientation-specific integration of gene cassettes, a nearby recombination site (attl), and a promoter, Pc, which ensures expression of the operon. There are three main classes of integrons based upon the type of integrase gene they possess: class 1 and class 2 integrons are the mot common, whereas class 3 are rare. Gene cassette Gene cassettes are genetic elements that may exist as free, circular, non-replicting DNA molecules when moving from one genetic site to another, but which are normally found as linear sequences that constitute part of a larger DNA molecule, such as a plasmid or bacterial chromosome. The genes carried on gene cassettes usually lack promoters and are expressed from a promoter on the integron. Bateriophage Bacteriophage (phage) are obligate intracellular parasites that multiply inside bacteria by making use of some or all of he host biosynthetic machinery (i.e., viruses that infect bacteria). They mediate the transfer of resistance genes. Salmonella spp. in swine – The abattoir as a link in the food chain 26 microorganism as Salmonella are part of a gene cassette inserted in an integron (Hall & Collis, 1998; Rowe-Magnus & Mazel, 2002). These gene cassettes contain genes conferring resistance to a range of antimicrobial agents, including aminoglycoside, betalactams, chloramphenicol and trimethoprim, as well as genes that confer resistance to antiseptics and disinfectants (Hall & Collis, 1998; Rowe-Magnus & Mazel, 2002). The evaluation of drug resistance at a molecular level is, therefore, an important tool for understanding the participation of genetic elements for the expression of resistance and its possible transfer among bacteria (Fluit, Visser, & Schmitz, 2001; Shahada et al., 2010; van Hoek et al., 2011). Moreover, in order to control the spread of resistance, is important to associate this evaluation with the study of the ecology of the environments in which spread is likely (van Hoek et al., 2011). SECTION II – Aims of the Thesis 27 SECTION II AIMS OF THE THESIS SECTION II – Aims of the Thesis 29 1. Aims of the thesis Salmonellae are accepted as an important source of human infections being mainly transmitted through food of animal origin, and pork products are recognized as a relevant source of salmonellosis (Berends et al., 1998a, 1998b; Boyen et al., 2008; De Busser et al., 2011; Delhalle, Saegerman, Farnir, et al., 2009; European Food Safety Authority, 2011b; Gebreyes et al., 2004; Valdezate et al., 2005). Particular MDR Salmonella clones mainly associated with asymptomatic swine and pork, have been increasingly involved in human infections (Dionisi et al., 2009; Hauser et al., 2010; Lucarelli et al., 2010; Switt, Soyer, Warnick, & Wiedmann, 2009). According to recent data, Portugal was found to be above the average EU-level of Salmonella prevalence both in breeding and production pig holdings and in slaughter pigs (European Food Safety Authority, 2008, 2009). In Portugal, swine production, abattoir stages and post-harvest pork meat production chain are insufficiently accessed (Antunes, Mourão, Pestana, & Peixe, 2011; Vieira-Pinto et al., 2005; Vieira-Pinto et al., 2006) and the contribution of slaughter and deboning operations in the contamination of meat with Salmonella remains to be studied. Furthermore, there is a lack of characterization of MDR clones in slaughtered swine. Additionally, in spite of the specificity of EU Food law applicable to abattoir and meat plants, there is a general lack of information about professional training for slaughterhouses and deboning room’s workers. The aim of this thesis was to fill the gaps mentioned above by increasing the knowledge about Salmonella in Portuguese slaughter swine and its impact in food chain and public health, enlightening meat handler’s participation in the overall process. Salmonella spp. in swine – The abattoir as a link in the food chain 30 Specific aims of the studies included were to: a) Identify the occurrence of Salmonella in slaughter swine from different abattoirs. b) Isolate Salmonella in different sources unveiling routes of cross-contamination in the abattoir environment, characterizing the genetic relatedness of the isolates. c) Investigate antimicrobial resistance in strains obtained from swine, carcasses, meat and meat handlers, characterizing resistance phenotypes. d) Identify genetic determinants of resistance, characterizing resistance genotypes. e) Evaluate and compare the level of general knowledge and practice in meat handlers from slaughter houses and meat plants, as a contribution to the comprehension of the participation of meat handlers in Salmonella MDR dissemination. This study was conducted in abattoirs and meat plants of the north region of Portugal, and the development of the experimental work and respective results are present in the following chapters under the form of research papers. SECTION III – Results 31 SECTION III RESULTS Salmonella spp. in swine – The abattoir as a link in the food chain 38 serotypes identified corresponded to single profiles (L1, G1, E1 and S1). The L1 PFGE type was identified in two consecutive carcasses (Nos.44 and 45) at abattoir D (Table 3). In 11.5% (95% CI 3.0–31.3) of the pigs that had Salmonellapositive lymph node samples, it was possible to identify the same genotype in other samples (carcass and/or meat) (Nos.35, 55 and 78) (Table 3). Regarding meat contamination, 28.6% (95%CI 9.6–58.0) of the positive meat samples belonged to Salmonella-positive carcasses where the same genotype was also identified (Nos. 35, 42, 77 and 78); 14.3% (95% CI 25.2–43.9) corresponded to Salmonella-positive pigs (Nos. 35, 78) where the same genotype was also identified in carcass and lymph node samples. Concerning meat handlers, Salmonella was isolated in 9.3% of the samples collected and 75% (95%CI 21.94–98.68) of positive meat handlers were in contact with positive meat samples, where the same genotype was isolated (Table 3). 4. Discussion In this study we observed a high Salmonella prevalence in slaughter swine and carcasses originating from multiple abattoirs supporting previous data (EFSA, 2008a; Vieira-Pinto et al., 2005). Importantly, we were also able to trace the occurrence of Salmonella along critical points in the slaughterhouse process. Salmonella was isolated in all abattoirs, and, with the exception of one abattoir (E), there were positive results from both lymph nodes and carcasses. It should be noted that despite the random selection of pigs, a common holding origin was used for the sampled animals in the different abattoirs (Table 3). Different Salmonella PFGE types were identified in the lymph nodes of these pigs, even when collected during the same time period, suggesting a diverse Salmonella population in the environment preceding the slaughterhouse (production level, transport and lairage) (Table 3). Contrary to what would be expected, albeit with marginal significance, abattoirs with the more hygienic scalding system had higher carcass contamination results when compared with those abattoirs using the less hygienic system (Table 1). This is probably due to other subsequent operations (such aspolishing and evisceration) as the pig carcasses pass through the flaming device, a contamination decreasing procedure, after scalding but prior to the referred operations. The contamination from the polishing operation can also be due to the equipment, in particular the flails and brushes of the polisher used during polishing, and the carcass splitter following polishing which should both be Table 3 Distribution of Salmonella clones among pig samples and meat handlers recovered in different abattoirs. Abbatoir Date Pig a Ileoceacal limph nodes Carcass Meat Meat handler A 06.08.07 1 1 S. Typh. T2 b A " 2 S. Derby D1 A " 3 S. Derby D1 A 24.09.07 7 S. Typh. T1 A " 8 S. 4,[5],12: i :- T1 A " 9 2 S. Typh. T1 A 14.05.08 93 1 S. Typh. T1 A 20.05.08 96 S. Typh. T1 A 28.08.08 99 2 S. Derby D1 A " 100 S. Typh. T2 B 16.10.07 13 S. 4,[5],12: i :- T1 B 30.10.07 17 S. Typh. T1 B " 18 3 S. Typh. T1 B 05.11.07 21 S. Typh. T1 B 08.11.07 22 S. Derby D1 B 27.11.07 26 S. Rissen R1 C 10.12.07 29 S. 4,[5],12: i :- T1 S. Typh. T5 C " 30 4 S. Typh. T3 S. Typh. T1 S. Typh. T3 C 12.12.07 31 S. Typh. T5 C " 35 4 S. Derby D1 S. Derby D1 S. Derby D1 C 18.12.07 36 S. Typh. T6 C " 37 S. Derby D1 D 21.01.08 41 5 S. Mbandaka M1 S. Typh. T3 S. Typh.T7 D " 42 S. Typh. T3 S. Typh. T3 D " 43 6 S. Typh. T3 S. Typh. T7 D 29.01.08 44 6 S. London L1 D " 45 S. Typh. T4 S. London L1 S. Rissen R1 D " 46 5 S. Typh. T4 E 08.02.08 47 S. London L1 F 13.02.08 51 S. Give G1 F 15.02.08 55 S. Derby D1 S. Derby D1 F " 56 S. Mbandaka M1 F 18.02.08 57 S. Typh. T9 F 20.02.08 60 S. Enteritidis E1 F " 61 S. Derby D1 G 05.03.08 67 7 S. Mbandaka M2 G 11.03.08 70 7 S. Rissen R1 S. Rissen R1 G 28.03.08 75 3 S. Sandiego S1 H 03.04.08 76 S. Typh. T8 c H 04.04.08 77 S. Typh. T8 c S. Typh. T8 c S. Typh. T8 c H " 78 S. Typh. T8 c S. Typh. T8 c S. Typh. T8 c H 18.04.08 86 8 S. Give G1 H 19.04.08 89 8 S. Typh. Ty4 a Superscript numbers 1,2,4,5,6,7,8, indicate pigs with same farm of origin. b S. Typhimurium and S. 4,5,12:i:- corresponds to genotypes T1–T9; S. Derby to D1; S. Rissen to R1; S. Mbandaka to M1,M2; S. London to L1; S. Give to G1; S. Enteritidis to E1; S. Sandiego to S1. c Corresponds to S. Typhimurium DT104. 85E. Gomes-Neves et al. / International Journal of Food Microbiology 157 (2012) 82–87 SECTION III – Results 39 disinfected with water at 82 °C between each carcass, however, the water used is frequently at a lower and ineffective temperature (Botteldoorn et al., 2004; De Busser et al., 2011; Lo Fo Wong et al., 2002; Vieira-Pinto et al., 2005). Abattoirs B and G, in spite of returning positive results from lymph node and carcass tests had no positive meat samples, probably resulting from better hygienic parameters and individual performance in the cutting and deboning rooms. However, in abattoir G, as with H, there is a causal link between the positive meat handler and the positive processed carcasses (Table 3). This study revealed high rates of occurrence in lymph nodes, similar to previous studies (EFSA, 2008a; Vieira-Pinto et al., 2005). Several authors refer to a correlation between the slaughter of asymptomatic Salmonella-carrier pigs and carcass and meat contamination (Berends et al., 1998b; De Busser et al., 2011; Delhalle et al., 2009; Vieira-Pinto et al., 2005, 2006). In our study, this association was only verified in three positive pigs carrying the same PFGE types in the lymph node and subsequent samples (carcass and/or meat) (Table 3). Furthermore, in Portugal there is a strong tradition of pork meat products that include the use of pork bowel in smoked and dried fermented sausages. In the abattoir, after evisceration and Official Veterinary Inspection, offal are washed and prepared in a separate room, including the removal of intestinal lymph nodes. This operation is a potential source of crosscontamination from the high percentage of Salmonella-positive, but consistently normal in appearance, lymph nodes. Besides the contribution to the complex cycle of contamination at slaughter house level, the raw bowel enters into the food chain through dispatch to butcher shops or meat plants where it is processed again, assuming a further role in Salmonella dissemination. The level of carcass contamination was above the EU baseline survey value and points to a hygiene problem, as EU food law imposes the surveillance of Salmonella in pig carcasses under criteria for process hygiene (Anonymous, 2005). In several consecutive carcasses, obtained from pigs which tested negative at lymph node level, the same PFGE type was identified, suggesting that the source of contamination was common and possibly environmental (such as from contact with equipment). This pattern was particularly frequent in abattoir D, with a high rate of pigs slaughtered per hour. In another case, a positive carcass was related to a positive lymph node sample from a previous animal, which contaminated contiguous carcasses in the slaughter line and then propagated the contamination to the cutting and deboning room, including the meat handler and several meat samples (Table 3). Fresh meat is not regularly tested for Salmonella contamination at meat plants as is required of minced meat or prepared meat by EU legislation (Anonymous, 2005). In this study the observed contamination of meat is much higher than the most recent published Portuguese data (EFSA, 2011) but nevertheless lower than other authors report (Delhalle et al., 2009). Moreover, strong evidence of crosscontamination between carcasses and meat is present, supporting the hypothesis that contamination is transferred between successive phases of pig slaughter and processing, as other authors have referred to (Berends et al., 1998b; Botteldoorn et al., 2004; De Busser et al., 2011; Lo Fo Wong et al., 2002; Swanenburg et al., 2001; Vieira-Pinto et al., 2005, 2006). In three of the Salmonella-positive meat handlers, the PFGE type coincided with that found on the carcasses and/or manipulated meat samples, as they were responsible for the cutting and deboning operations of the respective carcass (Table 3). This occurrence identifies a cross-contamination risk point and, again, a hygiene and public health problem, as besides the possibility of infection, workers could act as a vehicle for Salmonella transmission to the community. A previous study concerning the topic of cross contamination, carried out with the participation of meat handlers from these abattoirs, revealed that half of the respondents did not seem to be aware of the importance of changing clothes and working instruments, when they move from the tasks executed in ‘dirty spaces’(located at the abattoir) to ‘clean spaces’ (deboning room) in the same meat plant (Gomes-Neves et al., 2011). Although it is generally accepted that the hands of food handlers are an important vehicle for food cross contamination, the same study also observed that a high proportion of respondents from the group of meat handlers did not know all the required steps as part of a correct hand washing procedure. There was also a general lack of knowledge of microbiological food hazards, i.e. E. coli,Salmonella,Campylobacter and L. monocytogenes, and the related risks to their own and public health (Gomes-Neves et al., 2011). Our results regarding serotypes and their respective proportions are consistent with the Baseline EU survey and other studies of slaughtered pigs, where S. Typhimurium is the most frequent in all groups of samples, followed by S. Derby, S. Rissen and S. 4,[5],12:i:- (EFSA, 2008a, 2008b). The largest variation was noticed in the lymph nodes, where all of the serotypes were isolated except S. London (Table 2), which was not previously reported in Portuguese data. In the carcass and meat samples only four serotypes were isolated and the most prevalent were S. Typhimurium and S. Derby (Table 2), which is compatible with the EU survey results on carcass contamination, although Portugal was not included in that part of the study (EFSA, 2008a). In this study, different Salmonella PFGE types could be identified in samples from the same pig, in accordance with the results of other authors and unveiling cross-contamination critical points (Botteldoorn et al., 2004; De Busser et al., 2011). This finding contrasts another study, where it was shown that the same Salmonella genotype was identified in all positive samples from the same pig, with only one exception (Vieira-Pinto et al., 2006). Although there is causal linkage between Salmonella contamination in carcasses and lymph nodes,this pathof transmission is seen to be less relevant for this study when compared with other previous studies, underlining the importance of spreading contamination across carcasses, meat and meat handlers' hands, unveiling an important public health problem (Berends et al., 1998b; Botteldoorn et al., 2004; Vieira- Pinto et al., 2005, 2006). As other authors have previously noted, the main contamination source is probably a continuous contamination cycle between slaughtered pigs, the environment and the carcasses (Botteldoorn et al., 2004; De Busser et al., 2011; Swanenburg et al., 2001). Additionally, the dominant serotypes identified in this study are commonly associated with human disease (EFSA, 2011; King et al., S Braenderup H9812 Genetic Similarity index (%) 100 80 60 40 PFGE XbaI profiles . . . . . . . . . . . S1 .G1 . . . . . T1 T1 T4 T2 T3 T7 T6 T5 T9 T8 R1 D1 E1 M1 M2 L1 S 4,5:i: - (3) S Typhimurium (8) S Typhimurium (3) S Typhimurium (2) S Typhimurium (6) S Typhimurium (2) S Typhimurium (1) S Typhimurium (2) S Typhimurium (1) S Typh.DT104 (7) S Rissen (4) S Sandiego (1) S Give (2) S Derby (11) S Enteritidis (1) S Mbandaka (1) S Mbandaka (2) S London (3) Serotype (No. of strains) Fig. 1. Seventeen representative XbaI PFGE profiles of the 60 Salmonella isolates analyzed and their similarity dendogram (S. Braenderup H9812 was also restricted with XbaI and used as a size standard). 86 E. Gomes-Neves et al. / International Journal of Food Microbiology 157 (2012) 82–87 Salmonella spp. in swine – The abattoir as a link in the food chain 40 2011), including the monophasic variant S. 4,[5],12:i:- (5%) considered emergent in humans (Dionisi, et al., 2009; Lucarelli et al., 2010; Hauser et al., 2010; Switt et al., 2009) and S. Typhimurium DT104 (12%) (Antunes et al., 2006). Of particular concern is the presence of this strain on a meat handler, underlining the importance of the abattoir environment in spreading human-health threatening clones. In spite of the fact that the significance of the present results is limited in part by the sample size, this study indicates that pork meat is an important source of Salmonella and that abattoir procedures could promote its contamination. Although swine can harbor Salmonella before slaughter, the abattoir environment can contribute to further cross-contamination along the slaughter line, including contact with meat handlers. There has been a lack of recent data in Portugal concerning contamination of pork products by Salmonella and further studies should clarify and quantify this transference of contamination. The primary production phase and the slaughterhouse environment have been found to be the main sources of the contamination of carcasses (Berends et al., 1998a,1998b; Botteldoorn et al., 2004; De Busser et al., 2011; Delhalle et al., 2009; Vieira-Pinto et al., 2005, 2006) and, in order to improve standards in the post-harvest pork meat chain, measures have to be taken at these stages. Reducing the prevalence of Salmonella positive pigs at the primary production phase can significantly decrease one of the main sources of contamination at the abattoir (De Busser et al., 2011). However, the need to invest in general hygiene improvement, meat handlers' training, good manufacturing practice and HACCP implementation (Delhalle et al., 2009; Gomes-Neves et al., 2011; Lo Fo Wong et al., 2002), remains crucial to reducing cross-contamination and to maintain the level of contamination as low as possible in pork meat. Acknowledgments The authors would like to thank the staff and personnel of the participating abattoirs for their willingness to participate in the study and their cooperation during the sampling procedures, and in particular, Dr. Maria João Sousa. The authors gratefully acknowledge the skillful technical assistance provided by Maria José Mosqueiro, Maria Adelaide Paixão and Ana Cristina Santos. We are also grateful to the National Reference Laboratory of Antimicrobial Resistance, the Department of Infectious Diseases, and the National Health Institute Dr. Ricardo Jorge for their collaboration. References Anonymous, 2002. International Organization for Standardization, ISO 6579:2002. Microbiology of food and animal feeding stuffs—Horizontal method for the detection of Salmonella spp. Anonymous, 2005. Commission Regulation (EC) No 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs. Official Journal of the European Communities L 338, 1–25. Anonymous, 2006. Commission decision of 29 September 2006 concerning a financial contribution from the community towards a baseline survey on the prevalence of Salmonella in slaughter pigs to be carried out in the member states (notified under document number C(2006) 4306) (2006/668/EC). Antunes, P., Machado, J., Peixe, L., 2006. Characterization of antimicrobial resistance and class 1 and 2 Integrons in Salmonella enterica isolates from different sources in Portugal. Journal of Antimicrobial Chemotherapy 58, 297–304. Antunes, P., Mourão, J., Pestana, N., Peixe, L., 2011. Leakage of emerging clinically relevant multidrug-resistant Salmonella clones from pig farms. Journal of Antimicrobial Chemotherapy 66 (9), 2028–2032. http://dx.doi.org/10.1093/jac/dkr228. Berends, B.R., Van Knapen, F., Mossel, D.A., Burt, S.A., Snijders, J.M., 1998a. Impact on human health of Salmonella spp. on pork in The Netherlands and the anticipated effects of some proposed control strategies. International Journal of Food Microbiology 44, 219–229. Berends, B.R., Van Knapen, F., Mossel, D.A.A., Burt, S.A., Snijders, J.M., 1998b. Salmonella spp. on pork at cutting plants and at the retail level and the influence of particular risk factors. International Journal of Food Microbiology 44, 207–217. Botteldoorn, N., Herman, L., Rijpens, N., Heyndrickx, M., 2004. Phenotypic and molecular typing of Salmonella strains reveals different contamination sources in two commercial pig slaughterhouses. Applied and Environmental Microbiology 70 (9), 5305–5314. CDC (Centers for Diseases Control and Prevention), 2002. One-day (24–48 h) standardized laboratory protocol for molecular subtyping of Escherichia coli O157:H7, nontyphoidal Salmonella serotypes, and Shigella sonnei by Pulsed Field Gel Electrophoresis (PFGE). PulseNet PFGE Manual. CDC, Atlanta, Georgia, USA. De Busser, E.V., Maes, D., Houf, K., Dewulf, J., Imberechts, H., Bertrand, S., De Zutter, L., 2011. Detection and characterization of Salmonella in lairage, on pig carcasses and intestines in five slaughterhouses. International Journal of Food Microbiology 145, 279–286. Delhalle, L., Saegerman, C., Farnir, F., Korsak, N., Maes, D., Messens, W., De Sadeleer, L., De Zutter, L., Daube, G., 2009. Salmonella surveillance and control at post-harvest in the Belgian pork meat chain. Food Microbiology 26 (3), 265–271. Dionisi, A.M., Graziani, C., Lucarelli, C., Filetici, E., Villa, L., Owczarek, S., Caprioli, A., Luzzi, I., 2009. Molecular characterization of multidrug-resistant strains of Salmonella enterica serotype Typhimurium and monophasic variant (S. 4,[5],12: i:-) isolated from human infections in Italy. Foodborne Pathogens and Disease 6, 711–717. EFSA, 2006. Opinion of the scientific panel on biological hazards on the request from the commission related to “Risk assessment and mitigation options of Salmonella in pig production”. The European Food Safety Authority Journal 341, 1–131. EFSA, 2008a. Report of the task force on zoonoses data collection on the analysis of the baseline survey on the prevalence of Salmonella in slaughter pigs, part A. The European Food Safety Authority Journal 135, 1–111. EFSA, 2008b. Scientific opinion of the panel on biological hazards on a request from the European Commission on a quantitative microbiological risk assessment on Salmonella in meat: source attribution for human salmonellosis from meat. The European Food Safety Authority Journal 625, 1–32. EFSA, 2009. Analysis of the baseline survey on the prevalence of Salmonella in holdings with breeding pigs, in the EU, 2008, Part A: Salmonella prevalence estimates. The European Food Safety Authority Journal 7 (12). http://dx.doi.org/10.2903/ j.efsa.2009.1377 93 pp., Available online: www.efsa.europa.eu. EFSA, 2011. The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2009. The European Food Safety Authority Journal 9 (3), 2090. Fedorka-Cray, P.J., Gray, J.T., Wray, C., 2000. Salmonella infections in pigs. In: Wray, C., Wray, A. (Eds.), Salmonella in Domestic Animals. CAB International, Wallingford, pp. 191–207. Gebreyes, W.A., Thakur, S., Davies, P.R., Funk, J.A., Altier, C., 2004. Trends in antimicrobial resistance, phage types and integrons among Salmonella serotypes from pigs 1997–2000. Journal of Antimicrobial Chemotherapy 53, 997–1003. Gomes-Neves, E., Cardoso, C.S., Araújo, A.C., Costa, J.M.C., 2011. Meat handlers training in Portugal: a survey of knowledge and practice. Food Control 22, 501–507. Hauser, E., Tietze, E., Helmuth, R., Junker, E., Blank, K., Prager, R., Rabsch, W., Appel, B., Fruth, A., Malorny, B., 2010. Pork contaminated with Salmonella enterica Serovar 4, [5],12:i: an emerging health risk for humans. Applied and Environmental Microbiology 76 (14), 4601–4610. King, N., Lake, R., Campbell, D., 2011. Source attribution of nontyphoid salmonellosis in New Zealand using outbreak surveillance data. Journal of Food Protection 74 (3), 438–445. Lo Fo Wong, D.M., Hald, T., van Der Wolf, P.J., Swanenburg, M., 2002. Epidemiology and control measures for Salmonella in pigs and pork. Livestock Production Science 76 (3), 215–222. Lucarelli, C., Dionisi, A.M., Torpdahl, M., Villa, L., Graziani, C., Hopkins, K., Threlfall, J., Caprioli, A., Luzzi, I., 2010. Evidence for a second genomic island conferring multidrug resistance in a clonal group of strains of Salmonella enterica serovar Typhimurium and its monophasic variant circulating in Italy, Denmark, and the United Kingdom. Journal of Clinical Microbiology 48 (6), 2103–2109. Newcombe, R.G., 1998. Two-sided confidence intervals for the single proportion: comparison of seven methods. Statistics in Medicine 17, 857–872. Pritchett, L.C., Konkel, M.E., Gay, J.M., 2000. Identification of DT104 and U302 phage types among Salmonella enterica serotype Typhimurium isolates by PCR. Journal of Clinical Microbiology 38, 3484–3488. Soyer, Y., Moreno Switt, A., Davis, M.A., Maurer, J., McDonough, P.L., Schoonmaker-Bopp, D.J., Dumas, N.B., Root, T., Warnick, L.D., Gröhn, Y.T., Wiedmann, M., 2009. Salmonella enterica serotype 4,5,12:i:-, an emerging Salmonella serotype that represents multiple distinct clones. Journal of Clinical Microbiology 47 (11), 3546–3556. Swanenburg, M., Urlings, H.A.P., Snijders, J.M.A., Keuzenkamp, D.A., Van Knapen, F., 2001. Salmonella in slaughter pigs: prevalence, serotypes and critical control points during slaughter in two slaughterhouses. International Journal of Food Microbiology 70, 245–256. Switt, A.I., Soyer, Y., Warnick, L.D., Wiedmann, M., 2009. Emergence, distribution, and molecular and phenotypic characteristics of Salmonella enterica serotype 4,5,12:i. Foodborne Pathogens and Disease 6, 407–415. Tennant, S.M., Diallo, S., Levy, H., Livio, S., Sow, S.O., Tapia, M., Fields, P.I., Mikoleit, M., Tamboura, B., Kotloff, K.L., Nataro, J.P., Galen, J.E., Levine, M.M., 2010. Identification by PCR of non-typhoidal Salmonella enterica serovars associated with invasive infections among febrile patients in Mali. PLoS Neglected Tropical Diseases 4, 621. Valdezate, S., Vidal, A., Herrera-Leon, S., Pozo, J., Rubio, P., Usera, M.A., Carvajal, A., Echeita, M.A., 2005. Salmonella Derby clonal spread from pork. Emerging Infectious Diseases 11, 694–698. Vieira-Pinto, M., Themudo, P., Martins, C., 2005. Occurrence of Salmonella in the ileum, ileocolic lymph nodes, tonsils, mandibular lymph nodes and carcasses of pigs slaughtered for consumption. Journal of Veterinary. Medicine B 52, 476–481. Vieira-Pinto, M., Tenreiro, R., Martins, C., 2006. Unveiling contamination sources and dissemination routes of Salmonella sp. in pigs at a Portuguese slaughterhouse through macrorestriction profiling by pulsed-field gel electrophoresis. International Journal of Food Microbiology 110, 77–84. Wilson, E.B., 1927. Probable inference, the law of succession, and statistical inference. Journal of the American Statistical Association 22, 209–212. 87E. Gomes-Neves et al. / International Journal of Food Microbiology 157 (2012) 82–87 SECTION III – Results 41 Chapter 2. One health: Antimicrobial Resistance entering the food chain. Paper II. Clinically relevant multidrug resistant Salmonella enterica in swine, at slaughter: filling the gaps in the food chain. SECTION III – Results 43 Clinically relevant multidrug resistant Salmonella enterica in swine, at slaughter: filling the gaps in the food chain Authors: Eduarda Gomes-Neves, Patrícia Antunes, Vera Manageiro, Fátima Gärtner, Manuela Caniça, José Manuel Costa, Luísa Peixe Abstract The presence of clinically relevant Salmonella serotypes in slaughtered swine, carcasses, meat and meat handlers is scarcely studied. In this work, we characterized resistance phenotypes and genotypes in 60 Salmonella isolates from swine (lymph nodes, carcasses, meat) and meat handlers of Portuguese abattoirs (July 2007-August 2008). More than 50% of the isolates were resistant to tetracycline (T) [70%, tet(A)/tet(B)/tet(G)], streptomycin (S) [63%, aadA2/strA/strB], sulfamethoxazole (Sul) [62%, sul1/sul2/sul3] and ampicillin (A) [57%, blaPSE-1/blaTEM] and 37% carried class 1 integrons. Multidrug resistance was frequently observed in isolates (63%; n=38/60) from all samples and most of serotypes, including the ones frequently observed in human infections [S. Typhimurium (78%), S. 4,[5],12:i:- (75%), S. Derby (55%), S. Rissen (75%), S. London (75%)]. The S. 4,[5],12:i:- isolates mostly presented ASSuT phenotype [blaTEM/strA-strB/ sul2/tet(B)], typical of the European clone, being here firstly described with a ST phenotype [strA-strB-tet(A)-tet(B)]. Multidrug resistance [ANSSuT; blaTEM-strA-strB-sul2- tet(A)] in S. London was also firstly reported. The identification in slaughter swine and meat handlers’ samples of Salmonella serotypes carrying antibiotic resistance features similar to the previously characterized in clinically isolates provides the lacking food-chain evidences to link their transmission from animals to humans. The abattoir environment and the slaughter operations seem not only to maintain MDR serotypes originated from the pig reservoir, but also propagate them through cross-contamination processes, involving meat handlers. Keywords: Salmonella, antimicrobial agents, swine, pork meat, S. Typhimurium monophasic variant Salmonella spp. in swine – The abattoir as a link in the food chain 44 1. Introduction Ten to twenty percent of human Salmonella enterica infections in the EU may be attributable to pig sources, as reported by EFSA in 2010 [1]. In addition, an increasing antibiotic resistance trend has been consistently observed in pigs and pork products [2], which could reach humans through the food chain [1]. Particular multidrug-resistant (MDR) S. enterica isolates with clinical relevance, such as S. Typhimurium monophasic variant (S. 4,[5],12:i:-), S. Typhimurium DT104 and S. Rissen have been described in piggeries or in pork [3,4,5,6]. In spite of the evidence that the animal setting is a worldwide reservoir of MDR strains [2,5], their characterization in swine at slaughter, as well as in meat handlers’ samples, has been scarcely studied, a critical point to link swine as a source of human Salmonella infection cases [1,2,3]. Thus, in this work, we assessed the presence of clinically relevant multidrug resistant Salmonella serotypes in slaughtered swine, carcasses, meat and meat handlers from 8 abattoirs in Portugal. 2. Material and Methods 2.1. Salmonella isolates, serotypes and PFGE types A total of 60 S. enterica isolates (56 from swine: 26 ileoceacal limph nodes samples, 16 carcass swabs and 14 meat samples; 4 from meat handlers’ hands) collected between July 2007 and August 2008 from 8 abattoirs (A-H) (Table 1) were included in this study [7]. Briefly, isolates belonged to nine serotypes, which included 32 S. Typhimurium and 3 S. 4,[5],12:i:-, 11 S. Derby, 4 S. Rissen, 3 S. London, 3 S. Mbandaka, 2 S. Give, 1 S. Enteritidis and 1 S. Sandiego. After PFGE analysis those with a SD (Dice Band-based similarity coefficient) value > 85% were considered to belong to the same PFGE-type. As shown in Table 1, the S. enterica serotypes belonged to 17 PFGE types, nine being S. Typhimurium (T1-T9) (T1 includes S. 4,[5],12:i:- isolates), two S. Mbandaka (M1, M2) and one of each of the other 6 serotypes. SECTION III – Results 45 Table 1. PFGE types, antimicrobial resistance profiles and class 1 integrons of Salmonella serotypes isolated from swine and meat handlers Serotypea / PFGE typeb Abattoirc Sampled material Resistance phenotyped/ Class 1 integrons (gene cassettes) (number of isolates) (number of isolates) (number of isolates) Resistance genes profile (number of isolates) Typhimurium/T1 (8) A (4); B (3); C (1) Lymph node (3); Carcass (3); (AMP), (STR), (SUL), TET, (TMP)/ - Meat (2) (blaTEM), (strA-strB), (sul2-sul3), (tet(A)-tet(B)), 4,[5],12:i:-/T1 (3) A (1); B (1); C (1) Meat (1); Lymph node (2) (AMP), STR, (SUL), TET/ - (blaTEM), strA-strB, (sul2), (tet(A)), tet(B) Typhimurium/T2 (2) A (2) Lymph node (2) - 600 (1) Typhimurium/T3 (6) C (2); D (4) Lymph node (1); Carcass (3); AMP, (STR), (SUL), TET/ 400 (3) Meat (1); Meat handler (1) blaTEM, (sul3), (tet(A)), (tet(B)) Typhimurium/T4 (3) D (2); H (1) Lymph node AMP, STR, SUL, TET/ - blaTEM, (strA-strB), sul3, tet(B), (tet(G)) Typhimurium/T5 (2) C (2) Lymph node (1); Carcass (1) (AMP, TET)/ 600 (1) (blaTEM, tet(A), tet(B)) Typhimurium/T6 (1) C Meat AMP, CHL, STR, SUL, TMP/ - blaTEM, cmlA1, sul2 Typhimurium/T7 (2) D (2) Meat AMP, STR, SUL, TET / - blaTEM, sul3, tet(B) Typhimurium DT104/T8 (7) H (7) Lymph node (1); Carcass (2); AMP, CHL, STR, SUL, TET / 1000 (aadA2) + 1200 (blaPSE-1) (7) Meat (3); Meat handler (1) blaPSE-1, floR, aadA2, sul1, (tet(A)-tet(G)) Typhimurium/T9 (1) F Meat - - Derby/D1 (11) A(3); B(1); C(4); F(3) Lymph node (5); Carcass (4); (STR, SUL, TET) / 1000 (aadA2) (6) Meat (2) (aadA2, sul1, tet(A))e Rissen/R1 (4) B (1); D (1); G (2) Lymph node (1); Carcass (1); (AMP), (CHL), (STR), (SUL), TET, (TMP)/ 1000 (1); 2000 (dfrA12, orfF, Meat (1); Meat handler (1) (blaTEM), (cmlA1), (aadA2), (sul1-sul3), tet(A), (dfrA12) aadA2)(2) Mbandaka/M1 (1) G Lymph node STR/strA-strB - Mbandaka/M2 (2) D (1); F (1) Lymph node - - London/L1 (3) D (2); E (1) Carcass (2); Meat handler (1) (AMP, NAL, STR, SUL, TET) / 800 (1) (blaTEM, strA-strB, sul2, tet(A))f Give/G1 (2) F (1); H (1) Lymph node - - Enteritidis/E1 (1) F Lymph node NAL - Sandiego/S1 (1) G Lymph node - - aPCR assays for the identification of Salmonella enterica serotype Typhimurium DT104/U302 [4] and Salmonella enterica 4,[5],12:i:- were performed [6]. bClones are indicated by capital letters and a subindex (Typhimurium and S.4,[5],12:i:-, Ty1-Ty9; Mbandaka, M1-M2; Derby, D1; Rissen, R1; London, L1; Give, G1; Enteritidis, E1; Sandiego, S1) cAbattoirs are identified from A to H; dAMP, ampicilin; CHL, chloramphenicol, NAL, nalidixic acid; STR, streptomycin; SUL, sulfamethoxazole; TET, tetracycline; TMP, trimethoprim; Variable presence of a given resistance phenotype/ genotype among isolates belonging to the same PFGE-type appears between parenthesis; (-) Absence of antibiotic resistance. eThis resistance phenotype and genotype is present in 6 isolates out of 11. f This resistance phenotype and genotype is present in 2 isolates out of 3. Salmonella spp. in swine – The abattoir as a link in the food chain 46 2.2. Antimicrobial susceptibility testing All S. enterica isolates were tested for antimicrobial susceptibility by the disk diffusion method, following CLSI standards [8]. Ten antimicrobial agents were tested: ampicillin (A), gentamicin (G), kanamycin (K), streptomycin (S), ciprofloxacin (Ci), nalidixic acid (N), chloramphenicol (C), tetracycline (T), sulfamethoxazole (Su) and trimethoprim (W). Escherichia coli ATCC 25922 was the control strain. Ampicillin resistant isolates were further tested for susceptibility to extended-spectrum β-lactams (ceftazidime, ceftriaxone, cefotaxime, cefepime, cefoxitin, aztreonam and imipenem) and the double disk synergy test for ESBL detection was also performed [8]. An isolate was scored MDR if it had reduced susceptibility to three or more structurally unrelated antibiotics. 2.3. Characterization of antimicrobial resistance genes and class 1 integrons Genes coding for resistance to ampicillin (blaTEM, blaPSE-1, blaOXA-30), streptomycin (aadA, strA-strB), tetracycline [tet(A), tet(B), tet(G)], sulfamethoxazole (sul1, sul2, sul3), chloramphenicol (floR, cmlA, catA) and trimethoprim (dfrA1, dfrA12) were searched by PCR, using primers and conditions previously described [4,6]. The detection and characterization of class 1 integrons was also performed by PCR and sequencing as reported [4]. Positive and negative and controls were included in all PCR assays. 3. Results and discussion 3.1. Percentage of antibiotic resistance and resistance phenotypes Antibiotic resistance was found in 75% of all Salmonella isolates, 63% of which were MDR. Interestingly, lowest rates of resistance were observed in isolates from lymph nodes (58%) comparing to isolates from samples of subsequent slaughter operations (94% from carcasses and 86% from meat) or meat handlers (75%). These results suggest that the abattoir environment and the slaughter operations are contributing to the spread of antibiotic resistant Salmonella. Resistance to tetracycline (n=42; 70%), streptomycin (n=38; 63%), sulfamethoxazole (n=37; 62%) and ampicillin (n=34; 57%) was detected in a higher frequency than chloramphenicol (n=9; 15%), trimethoprim (n=5; 8%) and nalidixic acid (n=3; 5%), which may reflect the high usage of the first antibiotics in food-producing animals [9]. These resistance phenotypes were also the most frequently reported in Salmonella isolates from humans, pigs and pork meat in EU [2], in spite that overall in lower percentages than in our study. Susceptibility to extended-spectrum β-lactams and absence of ESBL-producing strains was found. The ASSuT (38%), ACSSuT (16%) and SSuT (13%) were the most SECTION III – Results 47 frequent resistance phenotypes among our isolates, which may provide a selective advantage in the intensive animal production setting. 3.2. Dissemination of resistance determinants and serotypes A diversity of resistance genes encoding resistance to different families of antibiotics was detected among isolates of different serotypes/clones and spread in different pig abattoirs and samples, as shown in Table 1. Thirty-seven percent of the isolates (n=22/60) were positive for class 1 integrons (400-2000 bp), namely S. Typhimurium (12/22), including all DT104 isolates 123 (7), S. Derby (6/22), S. Rissen (3/22) and S. London (1/22), a similar rate to the observed in Salmonella from pork food products collected in Portugal [4]. Moreover, these strains shared identical resistance determinants with previously national and international widespread serotypes/clones from human, pork food products, food producing animals and, particularly, swine, supporting their involvement in human infections [3,4,6,10,11,12]. S. Typhimurium (9 PFGE-types) identified in swine samples (6 abattoirs) and in meat handlers (2 abattoirs) presented high antimicrobial resistance (88%, n=28/32) and was mostly MDR (78%, n=25/32), supporting the trend described in other works for this serotype from other sources/niches [2,4,12,13]. The phenotype ASSuT was the most prevalent (47%, n=15/32) in this serotype, with some PFGE-types identified in diverse samples in the same abattoir, as a result of cross-contamination, and/or disseminated in different abattoirs (Table 1). Particularly, S. Typhimurium DT104 (T8) isolated in lymph nodes, carcasses, meat and a meat handler in a single abattoir, clearly related to cross-contamination originated from lymph node samples, presented the typical MDR pattern, previously identified in food, human and piggeries isolates [4,6], suggesting that slaughter operations play a role in the spread of such strains. S. 4,[5],12:i:- resistant to different antibiotics isolated from lymph nodes and in this study, firstly, in Portugal, in meat samples in 3 different abattoirs, indicates that pork food products is an important vehicle of this emergent serotype. These isolates presented 100% of clonal relatedness with S. Typhimurium (T1) recovered from the same abattoirs and were mostly associated with the R-type ASSuT [blaTEM/strA-strB/ sul2/tet(B)], typical of the “European clone” [3,5,6,13] of monophasic strains and previously characterized in Portuguese piggeries [6]. This data suggests that resistance determinants of both S. Typhimurium and its monophasic variant were maintained in the pig reservoir in spite of their genetic evolution [14]. Additionally, one 148 of the S. 4,[5],12:i:- isolates presented the R-type ST [strA-strB/tet(A)/tet(B)], to our knowledge here, firstly, reported, which may be considered in the surveillance of this emergent serotype. S. Rissen, other emergent serotype, revealed common resistance features [tetracycline-tet(A) and/or typical class 1 integron-dfrA12-orfF-aadA2] of this serotype [4,6,11]. Salmonella spp. in swine – The abattoir as a link in the food chain 54 card, it is necessary to attend 15 h of mandatory training on the following subjects: Meat Hygiene, Food Microbiology, Handlers’ Personal Hygiene, Working spaces and Equipments’Hygiene, Packaging of meat and meat products, Hygiene of meat selling and delivery, Food Safety and HACCP, Work Safety and Hygiene. However, this training and this card are not required for working in abattoirs and deboning rooms, where it is considered that the EU regulations No. 852/2004 and No. 853/2004 regulate the need for professional training. The Portuguese general law that regulates workconditions has a legal requirementof 35h of yearly training for all workers (Diário da República, 2003, 2004). Recently, much has been written specifically on training in the food industry, but a great part of it is rather specific in nature and has been limited to discussions on single segments, primarily hotels and restaurants (Barrows, 2000; Seaman & Eves, 2006). There is a general lack of information about professional training for slaughterhouses and deboning rooms’workers. The aim of this study was to evaluate and compare the level of general knowledge and practice of meat handlers from slaughterhouses and meat plants from northern Portugal, evaluating the professional training they have received. To our knowledge, this is the first survey on meat handling knowledge and practice in Portugal. Other similar studies have been reported in several countries focusing on food handlers (Gomes-Neves, Araújo, Ramos, & Cardoso, 2007; Jev� snik et al., 2008; Nel, Lues, Buys, & Venter, 2004; Seaman & Eves, 2006; Walker, Pritchard, & Forsythe, 2003). 2. Material and methods 2.1. Questionnaire design Theself-administeredquestionnaireused inthisstudycomprises 24 multiple choice questions with three or four possible answers, including “do not know”for the purpose of minimizing the possibility of selecting the correct answer by chance. In addition, the questionnaire has seven questions related to demographic and job characteristics of the respondents (age, gender, number of years of formal education, age at the beginning of professional activity, job description and years of experience in the present activity and present company, professional training and the opinion to additional training). The present questionnaire has been adapted from a questionnaire used in a previous study (Gomes-Neves et al., 2007). The questions were designed and structured in two groups. A group of questions designated “Knowledge”(14 questions) was intended to assess the respondent’s knowledge about HACCP, microbiologic hazards development, food poisoning and food borne illness, safety and health requirements, high-risk food groups, dirty and clean areas in the workspace and water temperature in knife sterilisers. A second group of questions designated “Practice”(10 questions) was designed to assess respondents’ habits focused on personal hygiene practice and cross contamination, working surfaces and instrument washing requirements and products, meat and chopped meat storage temperatures, freezing temperatures, temperature ranges and food poisoning agents development, water treatment and non-potable water use, as water supply and quality and food security and safety are intertwined (Kirby, Bartram, & Carr, 2003;Table 1). The participants answering the questionnaire have remained anonymous. Each participant has been informed of the purpose of the survey and that confidentiality would be assured. 2.2. Questionnaire delivery The questionnaire has been delivered inperson in seven red meat abattoirs with deboning rooms, during routine meat inspection of the Veterinary Official Services between May 2007 and May 2008, in two different regions of northern Portugal. In each meat plant, questionnaires have been delivered toall the employees performing tasks related with meat handling. The completed questionnaires have been collected in person one month later. 2.3. Statistical analysis The analysis of the questionnaires has been performed using the computer software SPSS  (SPSS Inc., Chicago, IL; version17.0). The significance of the statistical differences of the proportion of correct answers between the groups of participants classified according to professional training has been identified using the Chi-Square test. The 95% confidence intervals (95% CI) of the proportion of correct answers in each group have been estimated according to the Wilson procedure with a correction for continuity (Newcombe, 1998; Wilson, 1927). The differences in the mean scores of Knowledge and Practice questions between the same groups referred to above have been determined using one-way ANOVA with a post-hoc test. In all tests, the statistical significance was two-sided and considered significant at p<0.05. 3. Results 3.1. Quantitative results 3.1.1. Participants’response Answers have been obtained from all the meat plants contacted, but 10% of the employees have not returned the questionnaire. The number of participants was 159 (115 male and 44 female). All but one were Portuguese. The participants’general characteristics are presented in Table 2. 3.1.2. Comparative analysis of training areas and periods of time among participants Two different areas of professional training among meat handlers (MH) have been identified: 1. Good practice in food industry (GPFI), and 2. Work Safety and Hygiene (WSH). The vast majority of the respondents (72.7%) has had professional training. Table 1 Summary of the focus of the questionnaire contents. Questions “Knowledge” HACCP ewhat is it? Identify sterile food What happens to bacteria at 37  C? Food borne illness most frequent symptoms Food borne illness agents transmission Visual, olfactory or taste checks identify bacteria contaminated meat? Meat handler hygiene and health and food borne illness agents Health conditions that are not acceptable in food handling Potential health consequences of animal intestinal bacteria (E. coli,salmonella, Campylobacter and Yersinia) Listeria monocitogenes and food borne illness Dirty and Clean workspaces in the abattoir Food borne agents inactivation Temperature of knives sterilisers Questions “Practice” Working surfaces and instruments washing requirements and products Potable water use/water supply Red meat storage temperatures Chopped meat storage temperatures Freezing temperatures for meat Temperature ranges and food poisoning agents development Different situations that imply hand washing before food handling Different steps to correct hand wash Cross contamination and change of working instruments and clothes E. Gomes-Neves et al. / Food Control 22 (2011) 501e507502 SECTION III – Results 55 Twelve percent (12.03%) of the respondents have had training in GPFI (12.03%), 22.8% in WSH and 37.9% in both areas (BT). During the previous year, 37.7% of the MH have received between 20 and 35 h of training, but 24.7% have never attended professional training (NT). Eighteen percent have had more than 35 h of training. For comparison purposes, respondents were divided in four professional training groups: GPFI, WSH, BT (both training) and NT (no training). Fifty percent (50.3%) of MH with professional training think that training provides useful information to their work and 64.9% are interested in future training and consider it very important. 3.1.3. Comparative analysis of response to “Knowledge”and “Practice”questions The group of respondents that has had training in the two areas (BT) reached the highest mean score of proportion of correct answers in the group “Knowledge”(67.26 �21.05), followed by the GPFI with a mean score of 66.92 �16.36 correct answers; WSH had 49.21 �22.77 and NT 47.89 �22.63. In the group of questions “Practice”, GPFI has had the highest proportion of correct answers with a mean score of 70.53 �17.47, followed by BT (68.67 �22.58). The mean score of correct answers for WSH has been of 58.33 �19.93, and for NT 63.44 �21.70. The difference between the proportion of correct answers to the questions “Knowledge”and “Practice”is statistically significant between the groups (one-way ANOVA Table 3). For the group of questions “Knowledge”, a post-hoc test (Tukey HSD test) has defined two different homogenous groups, one with the respondents that have attended GPFI or both areas of professional training and the other with the respondents that have had WSH or no training. In the group of questions “Practice”, the same test has assumed two different groups, GPFI and NT. The other two groups (WSH and BT) could not be discriminated. This analysis underlines the fact that, for the questionnaire content and for the purpose of food safety improvement, WSH professional training has no positive impact. 3.2. Qualitative results It has been considered important to detect finer differences among the answers to questions that tested the quality of the information sought (Tables 4A and 4B). 3.2.1. “Knowledge”questions (Table 4A) 3.2.1.1. HACCP. Regarding HACCP, 29.3% of MH have never heard of the term and 7% are acquainted with the expression but do not know the meaning of it. Regarding training, from the WSH group, 55.6% answered “do not know”to the question “What is HACCP?”and that proportion increases to 66.7% in the NT group. The proportion of respondents who have given correct answers has been of 63.2% in the GPFI group and 51.7% in the BTgroup. This group has also had the highest proportion of incorrect answers: 31.7% (NT: 15.4%, WSH: 22.2% and GPFS:15,8%). These differences were statistically significant (p¼0.000 using Pearson Chi-Square test). 3.2.1.2. Food poisoning and food borne illness. Almost the half (47.4%) of GPFI, 58.3% of WSH, 53.3% of BT and 43.6% of NT believe that they can identify whether meat is contaminated with food poisoning bacteria by visual, olfactory or taste checks (p¼0.368, using Pearson Chi-Square test). Similar results have been obtained in other surveys among food handlers (Gomes-Neves et al., 2007; Jev� snik et al., 2008; Walker et al., 2003) The majority of the MH (60.1%) are aware that insects, other food handlers and raw food are sources of bacteria, but 26.3% of GPFI, 44.4% of WSH, 15% of BT and 33.3% think that MH can only contaminate meat if they are ill (p¼0.001, using Pearson Chi-Square test). Twenty six percent (26.3%) of GPFI, 30.6% of WSH, 11.7% of BT and 41.0% of NT believe that MH can only get sick if they have contact with animal blood during work activity (p¼0.000, using Pearson Chi-Square test). A significant majority of MH knows that diarrhoea is the symptom that is most associated with food borne illness (85.3%) but 33.3% of NT, 30.6% of WSH and 11.7% of BT have not been able to identify consequences of intestinal bacterial infection (E. coli,Salmonella, Campylobacter and Yersinia). These differences among groups of respondents were statistically significant (p¼0.001, using Pearson Chi-Square test). Sixty two percent (61.5%) of NT have answered “do not know”to the question that relates Listeria monocytogenes with food borne Illness and 55.6% of WSH, 38.3% of BT and 26.3% of GPFI have given the same answer. Sixteen (16.0%) percent of all MH knew the name of the bacteria but did not identify the disease or transmission paths (p¼0.108, using Pearson Chi-Square test). 3.2.1.3. Temperature and food poisoning agent’s inactivation. Twenty percent of WSH (19.5) and NT (20.4) have answered “do not know”to the question “What happens to bacteria at 37 �C?”. More than a half (52.6%) of GPFI, 41.7% of WSH, 51.7% of BT and 28.2% of NT think that pasteurised milk is a sterile product. Among the NTgroup, 43.6% have not answered the question “identify a sterile food product” (p¼0.105, using Pearson Chi-Square test). High temperature has been recognised as a safe method to destroy bacteria by 52.6% of GPFI, 50.0% of WSH, 56.7% of BT and 48.7% of NT but 24.4% of MH think that refrigeration also kills bacteria. The majority (64.6%) of MH knows that 82 �C is the correct temperature for the water in sterilisers for knives and steels in stations located along the slaughter floors (Eustace et al., 2007), but 21.1% of GPFI, 38.9% of WSH, 30.0% of BT and 28.2% of NT have answered incorrectly. The differences between the groups of respondents were not statistically significant. 3.2.1.4. Safety and health requirements. ManyMH didnotseemto be aware of basic safety and health requirements to work with food. A majority of GPFI, WSH and NT (52.6%, 52.8% and 51.3%, respectively) have not identified skin disease, gastrointestinal disturbances, eye/ Table 3 Percentage of correct answers to the “Knowledge”and “Practice”questions within each group defined by professional training. Participant group Question group Knowledge Practice N¼14 questions N¼10 questions GPFI (N¼36) 66.92 �16.36 a 70.53 �17.47 WSH (N¼19) 49.21 �22.77 58.33 �19.93 BT (N¼60) 67.26 �21.05 68.67 �22.58 NT (N¼39) 47.89 �22.63 63.44 �21.70 one-way ANOVA d.f. ¼3F¼10.393 p¼0.000 d.f. ¼3F¼3.986 p¼0.009 a Mean�1SD. Table 2 Demographic data and job information of the participants. Participants (N¼159) Average �SD Minimume maximum Age (N¼155) 35.19 �10.15 16e58 Years of formal education (N¼151) 6.50 �2.59 0e13 Age at the beginning of the professional activity (N¼153) 15.68 �2.53 9e24 Years of experience in the same activity (N¼133) 12.65 �9.35 0e35 Years in the present company (N¼154) 8.89 �7.57 0e33 E. Gomes-Neves et al. / Food Control 22 (2011) 501e507 503 Salmonella spp. in swine – The abattoir as a link in the food chain 56 ear and throat disease as conditions that are not acceptable in meat handling. Only 28.3% of BT ignored these conditions. Thirty four percent of the MH answered that only a skin disease is a non acceptable condition for meat handling. Sixty eight percent (67.5%) of the MH were aware of the need for skin injury protection in meat handling (p¼0.009, using Pearson Chi-Square test). According to Jacob (1989), routine medical examinations of food handlers are of little value because they merely reveal the health status of the worker at a specific point in time. The author further states that these medical examinations are unreliable and that carriers of pathogens are unlikely to transmit theseorganisms. In this study, 72.4% of the respondentshave indicated that they have been to routine medical examinations during the previous year, while 5.9% indicated that they have gone because they felt sick, whereas 12.5% needed to undergo medical examinations before employment. Food handlers must undergo medical examinations before employment to assess the general health. However, it has been suggested that routine medical examinations are regarded as not being cost-effec- tive and, in fact, unreliable (Jacob, 1989; Nel et al., 2004). 3.2.1.5. Dirty and clean workspaces at the abattoir. Sixteen percent (15.8%) of GPFI, 44.4% of WSH, 20.0% of BT and 35.9% of NT have identified incorrectly all the dirty areas in the abattoir. Of all MH, 10% think that only the lairage is a dirty space, and 18% have only identified the room where offal are washed and prepared (p¼0.001, using Pearson Chi-Square test). 3.2.2. “Practice”questions (Table 4B) 3.2.2.1. Instruments and working surface cleaning. Eighty nine percent (88.5%) of the respondents were aware of the working surfaces and instruments washing and disinfection routine and correct steps and only 5.7% answered that they did not have contact with that operation. As far as disinfection is concerned, 25.3% of MH thought that sodium hypochlorite is the best disinfectant in meat industry but 47.4% were aware of the need for regular rotation of products for this purpose (Meyer, 2006). However, 12% did not know that, after the use of disinfectant on instruments and surfaces, both of them must be cleaned with potable water. Forty two percent (42.1%) of GPFI, 25.0% of WSH, 31.7% of BT and 30.8% of NT thought that non-potable water could be used for the cleaning of working surfaces and instruments. These differences were not statistically significant. 3.2.2.2. Personal hygiene. To the question “When do you wash your hands during a work day”only 3.2% of MH have not answered and 89.2% have answered that they washes them several times and whenever the activity is interrupted (p¼0.181, using Pearson Chi- Square test). To the question “different steps to correct hand wash”, 5.8% of MH have not answered. The majority of MH referred all the steps for a correct hand wash, however 21.1% of GPFI, 38.9% of WSH, 30.0% of BT and 43.6% of NT have answered incorrectly, because they have not mentioned the use of nail brush (p¼0.015, using Pearson Chi-Square test). Table 4B Percentage of correct answers and 95% Confidence Intervals a (CI) of the questions “Practice”(qualitative results). Questions “Practice”% Correct Answers (95% CI) GPF WSH BT NT N¼36 N¼19 N¼60 N¼39 Working surfaces and instruments washing 84.2 (59.5e95.8) 94.4 (80.0e99.0) 90.0 (78.8e95.9) 89.7 (74.8e96.7) Working surfaces and instruments disinfection products 47.4 (25.2e70.5) 36.1 (21.3e53.8) 58.3 (44.9e70.7) 43.6 (28.2e60.2) Potable water use for washing purposes 57.9 (34.0e78.9) 58.3 (40.9e74.0) 61.7 (48.2e73.6) 59.0 (42.2e74.0) Temperature ranges and meat preservation 36.8 (17.2e61.4) 27.8 (14.8e45.4) 38.3 (26.3e51.8) 25.6 (13.6e42.4) Red Meat storage temperatures 100.0 (79.1e100.0) 63.9 (46.2e78.7) 83.3 (71.0e91.3) 74.4 (57.6e86.4) Chopped meat storage temperatures 68.4 (43.5e86.4) 38.9 (23.6e56.5) 56.7 (43.3e69.2) 43.6 (28.2e60.2) Freezing temperatures for meat 57,9 (34.0e78.9) 47,2 (30.8e64.3) 56,7 (43.3e69.2) 41,0 (26.0e57.8) Different situations that imply hand washing before meat handling 100.0 (79.1e100.0) 100.0 (88.0e100.0) 86.7 (74.9e93.7) 84.6 (68.8e93.6) Different steps to correct hand wash 78.9 (53.9e93.0) 52.8 (35.7e69.2) 70.0 (56.6e80.8) 43.6 (28.2e60.2) Cross contamination and change of working instruments and clothes 21.0 (7.0e46.1) 27.8 (14.8e45.4) 31.7 (20.6e45.1) 33.3 (19.6e50.3) a Wilson procedure with a correction for continuity (Newcombe, 1998; Wilson, 1927). Table 4A Percentage of correct answers and 95% Confidence Intervals a (CI) of the questions “Knowledge”(qualitative results). Questions “Knowledge”% of Correct Answers (95% CI) GPF WSH BT NT N]36I N¼19 N¼60 N¼39 What is HACCP? 63.2 (38.6e82.8) 22.2 (10.7e39.6) 51.7 (38.5e64.6) 17.9 (8.1e34.1) Identify sterile food 21.1 (7,0e46.1) 25.0 (12.7e42.5) 31.7 (20.6e45.1) 28.2 (15.6e45.1) What happens to bacteria at 37 � C? 89.5 (65.5e98.2) 61.1 (43.5e76.4) 83.3 (71.0e91.3) 59.0 (42.2e74.0) Food borne illness most frequent symptoms 100.0 (79.1e100.0) 77.8 (60.4e89.3) 95.0 (85.2e98.7) 71.8 (54.9e84.4) Food borne illness agents transmission 73.7 (48.6e89.9) 52.8 (35.7e69.2) 65.0 (51.5e76.5) 56.4 (39.8e71.8) Visual, olfactory or taste checks identify bacteria contaminated food? 42.1 (21.1e66.0) 41.7 (26.0e59.1) 45.0 (32.3e58.3) 51.3 (35.0e67.3) How can MH contaminate meat? 73.7 (48.6e89.9) 55.6 (38.3e71.7) 85.0 (72.9e92.5) 56.4 (39.8e71.8) MH can get ill in consequence of meat handling? 47.4 (25.2e70.5) 63.9 (46.2e78.7) 88.3 (76.8e94.8) 51.3 (35.0e67.3) Health conditions that are not acceptable in food handling 47.4 (25.2e70.5) 36.1 (21.3e53.8) 65.0 (51.5e76.5) 30.8 (17.5e47.7) Potential health consequences of animal intestinal bacteria 100.0 (79.1e100.0) 58.3 (40.9e74.0) 75.0 (61.9e84.9) 43.6 (28.2e60.2) Listeria monocitogenes and food borne illness 68.4 (43.5e86.4) 36.1 (21.3e53.8) 53.3 (40.1e66.1) 33.3 (19.6e50.3) Dirty and Clean workspaces in the abattoir 78.9 (53.9e93.0) 52.8 (35.7e69.2) 80.0 (67.3e88.8) 48.7 (32.7e65.0) Food borne agents inactivation 52.6 (29.5e74.8) 50.0 (33.2e66.8) 56.7 (43.3e69.2) 48.7 (32.7e65.0) Temperature of knives sterilisers 78.9 (53.9e93.0) 55.6 (38.3e71.7) 66.7 (53.2e78.0) 59.0 (42.2e74.0) a Wilson procedure with a correction for continuity (Newcombe, 1998; Wilson, 1927). E. Gomes-Neves et al. / Food Control 22 (2011) 501e507504 SECTION III – Results 57 3.2.2.3. Temperature control. From the three ranges of temperatures presented, 0e4�C/5e65 �C/70e80 �C, only 32.3% of the MH identified the range of 5e65 �C as the high-risk meat storing temperature. The GPFI group has also had the highest proportion of incorrect answers (63.2%), followed by BT (53.3%), WSH (52.8%) and NT (51.3%). Interestingly, the GPFI group seems to be confident regarding this topic since none of the respondents report “do not know”to this question, although the majority of the subjects has answered incorrectly. Seventy nine percent (78.6%) knew of the correct red meat storage temperature but only half of MH have reported the correct freezing temperature (50.6%) and the correct storage temperature for chopped meat (51.3%). If we consider professional training, WSH group has had a lower proportion of correct answers on red meat storage temperature (63.9%) than NT (74.4%). Twenty six percent (26.3%) of GPFI, 44.4% of WSH, 30.0% of BT and 23.1% of NT have answered incorrectly to the question about chopped meat storage temperature and 33.3% of NT answered “do not know”(p¼0.036, using Pearson Chi-Square test). 3.2.2.4. Change of clothes and instruments and cross contamination sources. Only twenty one percent (21.1%) of the GPFI, 27.8% of the WSH, 31.7% of BT and 33.3% of NT recognise the need to change clothes and knives by the end of the work at the abattoir (mainly in the first hours of the day), when they continue their tasks in the deboning room of the same building (p¼0.087, using Pearson Chi- Square test). Fifty seven percent (56.8%) of all MH recognise the need to change their protective clothing but do not admit the importance of replacing knives and 5.8% answered that is correct to carry their clothes and knives from the slaughter room into the deboning room. Regarding the porosity of surfaces, it can be observed that porous surfaces (clothes, aprons, sponges, etc.) show lower transfer rates when compared to non-porous surfaces as stainless steel and knobs (Kusumaningrum, Van Putten, Rombouts, & Beumer, 2002; Scott & Bloomfield, 1990). However, in this case, although apparently a lower risk might be associated to transfer from fabrics, it should be noted that the residual water (and eventually blood) accumulated in clothes would enable bacteria to survive for longer periods and, consequently, bacterial transfer events could also be prolonged (Bloomfield et al., 1994; Eustace et al., 2007; Rusin, Maxwell, & Gerba, 2002). In addition to protective clothing fulfilling a safety function, 44.7% wear stainless steel mesh gloves. Stainless steel gloves also require cleaning and sterilisation, but these gloves are difficult to clean, due to their woven construction (Van Zyl,1998). Upon asking the respondents about the frequency of cleaning, 59.5% have reportedthat they wash and sterilise their glovesseveral times a day, whenever they are visibly dirty (usually full of fatty or bloody deposits). Furthermore, a small percentage, 11.1% sterilises their gloves on a daily basis (end of work), while 22.2% have answered they never washed or sterilised their gloves because they were not connected with cleaning tasks. According to the Canadian Food Inspection Agency (CFIA), these gloves should be sterilised at regular intervals throughout the working shifts to prevent cross contamination between gloves and meat (CFIA, 1990; Nel et al., 2004). On the matter of Pre-Requisite Plans (PRP) participation, 56.6% did not participate in any activity. The highest participation is related with cleaning activity, since 17.8% complete cleaning checklist forms and only 9.2% participate in meat temperature control activities, whereas 8.6% have maintenance related tasks. 4. Discussion The questionnaire designed for the present study has allowed to detect quantitative differences in “knowledge”and “practice”skills among the participants. The satisfactory participation has permitted to highlight the existence of differences between MH who have and have not received professional training, obtaining the groups NT, WSH, GPFI and BT. This is remarkable and somewhat reassuring. Nevertheless, a further finer analysis of the content of the questions themselves (qualitative results) has not led to the same sense of reassurance. The proportion of correct answers in the MH groups who have had GPFI or BT training is significantly higher than the others from a statistical point of view, but results have also indicated that WSH training is not relevant to Food Hygiene and Food Safety knowledge and practice. Regarding HACCP, which is a recent and relevant imposition of the EU Food Law, there was still a high proportion of MH (even with professional training, the WSH group) who were unacquainted with the concept. To the question “What is HACCP”, only half of BT have answered correctly and this group has also had the highest proportion of incorrect answers, somehow contrary to what should be expected. It seems to be very difficult to implement an HACCP based system in this industry, when a high proportion of employees is not familiar with this reality and does not participate in PRP. Mortimore and Smith (1998) have shown that many trainers had been willing to provide HACCP training without considering the scope (what has to be taught and what need not) and the depth of coverage. Although numerous companies have developed, documented and implemented training programs, few understand why employee training is important, what their training requirements are, orhow to assess the effectiveness of in-house training programs. In the matter of meat storage temperatures, e.g. red meat, the WSH group has had the highest rate of incorrect answers and the lowest of correct answers. The BT group has not had better results, regarding the fact that they associate two different areas of professional training. A high proportion of GPFI, WSH and BTrely on visual, olfactory or taste checks to identify bacteria contaminated meat. This finding is difficult to explain, considering that they all have had professional training. The study demonstrates that there is also a general lack of knowledge on microbiological food hazards, i.e. E. coli,Salmonella,Campylobacter,Yersinia and L. monocytogenes. It is generally accepted that the hands of food handlers are an important vehicle of food cross contamination and that improved personal hygiene and scrupulous hand washing lead to the basic control of spread of potentially pathogenic transient microorganisms (Allwood, Jenkins, Paulus, Johnson, & Hedberg, 2004; Daniels et al., 2002;Fry, Braden, Griffin, & Hughes, 2005;Lues & Van Tonder, 2007; Sneed, Strohbehn, Gilmore, & Mendonca, 2004). In this study, it has been possible to observe that in the four groups there are respondents who do not know all the steps for a correct hand wash. According to the results of Shojaei, Shooshtaripoor, and Amiri (2006), a dramatic reduction in hand contamination has been observed after a simple intervention that included a face-to-face health education on strict hand washing after visiting the toilet. Concerning the topic of cross contamination, the majority of MH does not seem to be aware of the importance of changing clothes and working instruments, when they move from the tasks developed in “dirty spaces”(located at the abattoir) to “clean spaces”(deboning room). In addition, they also seem to have difficulties in identifying the differences between the spaces themselves. The UK surveillance system has reported that cross contamination was the main contributing factor (32%) for theoutbreaks investigated in the period of 1999e2000 (WHO, 2003). Similarly, the US Centres for Disease Control and Prevention (CDC) have reported that 18 and 19% of food borne diseases caused by bacteria in the years 1993 and 1997 in the United States were associated with contaminated equipment and poorhygienepractices,respectively(CDC, 2000). Moreover, although most outbreaks result from extensive growth at abusive storage temperatures, insufficient cooking, etc., many are also associated with bacterial cross contamination/recontamination (Notermans, E. Gomes-Neves et al. / Food Control 22 (2011) 501e507 505 Salmonella spp. in swine – The abattoir as a link in the food chain 58 Zwietering, & Mead, 1994; Roberts, 1990). Similarly, various authors have stated that cross contamination of bacterial and viral pathogens in homes and in food-service establishments could well be the major contributing factor to sporadic and epidemic food borne illnesses (Beumer & Kusumaningrum, 2003; Bloomfield, 2003; Chen, Jackson, Chea, & Schaffner, 2001). In the present study, a high proportion of respondents admits a potentially dangerous behaviour on a daily basis, as 56.8% (n¼88) recognise theneed to change their clothes but do not admit the importance of changing knives when they end the work atthe abattoir andstart atthe deboning room. In a HACCP based system perspective this is an unacceptable occurrence. As a result of EU law implementation, Portuguese slaughterhouse and deboning room owners need to offer professional training to their employees but they do not show special concerns about their own training program and its contents. According to the evaluation of the present study, in a high proportion of MH who have had professional training in WSH, this training has not produced a significant contribution to meat safety. Furthermore, as several authors suggested, it seems that most managers in food and meat industry have a limited understanding of the global food safety strategy (Ehiri, Morris, & McEwen, 1997; Khandke & Mayes, 1998; Mortimore & Smith, 1998; Williams et al., 2003). MacAuslan (2003) has pointed out that the majority of food businesses do not have satisfactory training policies for all their staff. The authoremphasized that too much reliance is being placed upon attaining a training certificate rather than attention paid to achieving competency in food hygiene practice. More emphasis and resources need to be diverted towards assisting managers to become highly motivated to food hygiene managers who develop and maintain a food safety background within their business. Few employers perceive a relationship between investment in their human resource assets and successful business performance, and training is often undertaken only to meet perceived statutory or inspection requirements (Pratten & Curtis, 2002; Seaman & Eves, 2006). Food business owners may be tempted to place the burden of training responsibility on an external employer, and not shoulder any responsibility themselves. This problem has two sides; firstly the employer lacks key management skills in leadership, motivation, training and evaluation, and secondly going for a certificate course as it is the “done thing”(MacAuslan, 2003). What we have observed in the present study is that the pressure to accomplish the law leads employers to get specialised training for their employees; however, there is no evidence that the worker practices improve when training programs provide only information (Nieto-Montenegro, Brown, & LaBorde, 2008; Rennie, 1994). Several studies have demonstrated that increasing knowledge does not necessarily lead to changes in behaviours (Clayton, Griffith, Price, & Peters, 2002; Ehiri et al., 1997; Rennie, 1994, 1995). To be effective, training programs should be based on appropriate adult education theory (Rhodes,1988). In the present study, we have verified a low educational level of MH, the average formal education years being 6.5 (in Portugal the mandatory formal education takes 12 years) in a group with a mean age of 35 (Table 2), which may be a possible explanation factor for our results. The findings in the study of Toh and Birchenough (2000) affirmed education as an important link to the two variables: knowledge and attitudes; customs and environment. Some other authors suggest thatthe training programs should incorporate activities that support skills development relevant to real life situations in which the workers can put information into practice (Edmunds, Lowe, Murray, & Seymour, 1999; Kowalski & Vaught, 2002). Food hygiene training is a legal requirement within food industry and should be only one part of an effective food safety management strategy. Training will only lead to an improvement in food safety if the knowledge imparted leads to desired changes in behaviour at the workplace (Nieto-Montenegro et al., 2008; Seaman & Eves, 2006). To our knowledge, professional training of MH in Portugal has been “classroom based”and this study aims to contribute to a reflexion on the need for evaluation towards practical improvements. Evidence from the literature suggests that food hygiene training as a mean of improving food safety standards is limited by a lack of understanding of those factors contributing to successful outcomes. Training activities closely associated with work environment would be more appropriate than food hygiene courses that operate divorced from the workplace and use solely knowledge-based assessment techniques (Seaman & Eves, 2006). The training of managers is a necessary precursor to the implementation of realistic food safety practices within the workplace. The effectiveness of training is very dependent on both management attitude and their willingness to provide the resources and systems for food handlers to implement good practices. There is a need to develop training methods that proved to change behaviour as well as imparting knowledge (Egan et al., 2007). Further research in issues including course content, training location, duration of courses, motivational factors and refreshment training is needed. Such research needs to be clearly thought out, well designed with good baseline data to achieve worthwhile results (Egan et al., 2007; Seaman & Eves, 2006). Seaman (2010) proposes the Food Hygiene Training Model which includes evaluation stages, managerial components and overall performance measures to take into account both the effective planning of the training program, the managerial support required to facilitate the training process, and the overall performance measures needed to ensure that training transfers into the required safe food handling behaviours. The proposed model incorporates three evaluation stages of the food handlers: 1) documented training needs with individual record, establishing a starting point; 2) knowledge test and/or practical skill assessment shortly after training, assessing any deficiencies in skills or knowledge at this stage; 3) food handlers evaluation of the training program to measure the perceived value and relevance of the training program, allowing respondents to portray approval or disapproval towards certain aspects of the training (Seaman, 2010). The overall performance measures include two final evaluation categories: the effect of food hygiene training on the individual food handler and the effect on the organization (Seaman, 2010). The success of training relies on the choice of the program, considering the relevance of the course to work activities, and providing food hygiene training in a language and at a level that allows the food handler to understand the content (Rennie, 1994; Seaman, 2010). Authors suggest that food hygiene courses should be shorter and focused on the needs and motivation of the participant, and include refresher training to provide both a physical and psychological environment conductive to food handler development and the enactment of safe food handling practices (MacAuslan, 2003; Rennie, 1994; Seaman, 2010; Worsfold, 2001). The significance of the present results is limited in part by the sample size and by the fact that it has based on self-reported behaviour and practice. It is possible to conclude, however, that EU regulations have had a positive outcome in the matter of professional training of MH in Portugal. Operators, however, cannot rely on the fact that training has ever taken place. They must assume that all employees will need thorough, repeated training in the area of food hygiene and safety, as we observed that WSH training is not relevant to this aim (in spite of being relevant in terms of occupational safety and health). We suggest what can be a major concern in the moment of hiring new employees: to assess knowledge in food safety and promote immediate professional training, in addition to asking about previous work experience. In the present study, the MH show an average of 12.6 years of experience in the activity. However, the respondents have had poor results on the HACCP, microbiological hazards, temperature control, personal hygiene and cross contamination subjects. E. Gomes-Neves et al. / Food Control 22 (2011) 501e507506 SECTION III – Results 59 In this activity, characterized by hard physical work and a traditionally low educational level of the workers, professional training should be adapted, with a strong connection knowledge-practice, considering motivational factors and beliefs. Behaviour changes in MH should be evaluated according to those conditions, encouraging the learning process and rewarding practical improvements. Acknowledgments We gratefully acknowledge to Prof. Fátima Gärtner, Prof. Margarida Fonseca Cardoso and Dr. Maria do Céu Cláudio for their comments and help in the preparation of the manuscript and Dr. Ana Isabel Oliveira for her cooperation on questionnaire delivery. References Allwood, P. B., Jenkins, T., Paulus, C., Johnson, L., & Hedberg, C. W. (2004). Hand washing compliance among retail food establishment workers in Minnesota. Journal of Food Protection, 67(12), 2825e2828. Barrows, C. W. (2000). An exploratory study of food and beverage training in private clubs. International Journal of Contemporary Hospitality, 12(3), 190e197. Beumer, R. R., & Kusumaningrum, H. (2003). Kitchen hygiene in daily life. International Biodeterioration & Biodegradation, 51, 299e302. Bloomfield, S. F. (2003). Home hygiene: a risk approach. International Journal of Hygiene and Environmental Health, 206, 1e8. Bloomfield, S. F., Arthur, M., Van Klingeren, B., Holah, J. T., Pullen, W., & Elton, R. (1994). An evaluation of the repeatability and reproducibility of a surface test for the activity of disinfectants. Journal of Applied Bacteriology, 76, 86e94. Canadian Food Inspection Agency. (1990). Meat hygiene manual of procedures, Canada. Available at: www.inspection.gc.ca Accessed 26.03.02. CDC (Center for Disease Control and Prevention). (2000). Surveillance for foodborne disease outbreaks e United States,1993e1997. Available from. http://www.cdc.gov/. Chen, Y., Jackson, K. M., Chea, F. P., & Schaffner, D. W. (2001). Quantification and variability analysis of bacterial cross-contamination rates in common food service tasks. Journal of Food Protection, 64, 72e80. Clayton, D. A., Griffith, C. J., Price, P., & Peters, A. C. (2002). Foodhandlers’beliefs and self-reported practices. International Journal of Environmental Health Research, 12(1), 25e29. Daniels, N. A., MacKinnon, L., Rowe, S. M., Bean, N. H., Griffin, P. M., & Mead, P. S. (2002). Food borne disease outbreaks in United States schools. The Pediatric Infectious Disease Journal, 21(7), 623e628. Diário da República. (1998). 1 a série A, N  65. Decreto-Lei n  . 67/98 de 18 de Março. Diário da República I, Série A(no. 65). Diário da República. (2003). 1 a série A, N  197, Lei no 99/2003 de 27 de Agosto. Aprova o Código do Trabalho. Diário da República, 1 a série A(197), 5558e5675. Diário da República. (2004). 1 a série A, No 177, Lei N  35/2004 de 29 de Julho. Regulamenta a Lei No 99/2003, 1 a série A(177), 4810e4885. Diário da República. (2006). 1 a série No 146 Dec-lei 147/2006 31 Julho 2006. Diário da República, 1 a série(146), 5442e5451. Edmunds, C., Lowe, K., Murray, M., & Seymour, A. (1999). The ultimate educator: Achieving maximum adult learning through training and instruction. Office for- Victims of Crime. Website address: http://www.ojp.usdoj.gov/ovc/assist/ educator/welcome.html. Egan, M. B., Raats, M. M., Grubb, S. M., Eves, A., Lumbers, M. L., Dean, M. S., et al. (2007). A review of food safety and food hygiene training studies in the commercial sector. Food Control, 18, 1180e1190. Ehiri, J. E., Morris, G. P., & McEwen, J. (1997). Evaluation of a food hygiene training course in Scotland. Food Control, 8(3), 137e147. Eustace, I., Midgley, J., Giarrusso, C., Laurent, C., Jenson, I., & Sumner, J. (2007). An alternative process for cleaning knives used on meat slaughter floors International. Journal of Food Microbiology, 113, 23e27. Fry, A. M., Braden, C. R., Griffin, P. M., & Hughes, J. M. (2005). Foodborne disease. In G. L. Mandell, J. E. Bennett, & R. Dolin (Eds.), Principles and practice of infectious diseases (6th ed.). (pp. 1286e1297) New York: Elsevier, Churchill Livingston. Gomes-Neves, E., Araújo, A. C., Ramos, E., & Cardoso, C. S. (2007). Food handling: comparative analysis of general knowledge and practice in three relevant groups in Portugal. Food Control, 18(6), 707e712. Jacob, M. (1989). Safe food handling: A training guide for managers of food service establishments. Geneva: World Health Organization. Jev� snik, M., Hlebec, V., & Raspor, P. (2008). Food safety knowledge and practices among food handlers in Slovenia. Food Control, 19, 1107e1111. Khandke, S. S., & Mayes, T. (1998). HACCP implementation: a practical guide to the HACCP plan. Food Control, 9(2e3), 103e109. Kirby, R. M., Bartram, J., & Carr, R. (2003). Water in food production and processing: quantity and quality concerns. Food Control, 14(5), 283e299. Kowalski, K. M., & Vaught, C. (2002). Principles of adult learning: application for mine trainers. National Institute for Occupational Safety and Health Information Circular, 946, 3e8. Kusumaningrum, H. D., Van Putten, M. M., Rombouts, F. M., & Beumer, R. R. (2002). Effects of antibacterial dishwashing liquid on foodborne pathogens and competitive microorganisms in kitchen sponges. Journal of Food Protection, 65, 61e65. Legnani, P., Leoni, E., Berveglieri, M., Mirolo, G., & Alvaro, N. (2004). Hygienic control of mass catering establishments, microbiological monitoring of food and equipment. Food Control, 15, 205e211. Lues, J. F. R., & Van Tonder, I. (2007). The occurrence of indicator bacteria on hands and aprons of food handlers in delicatessen sections of a retail group. Food Control, 18, 326e332. MacAuslan, E.(2003). The boss, the owner, the proprietor. The food hygiene manager? The Journal of the Royal Society for the Promotion of Health, 123(4), 229e232. Martínez-Tomé, M., Vera, A. M., & Murcia, A. (2000). Improving the control of food production in catering establishments with particular reference to the safety of salads. Food Control, 11, 437e445. Meyer, B. (2006). Does microbial resistance to biocides create a hazard to food hygiene? International Journal of Food Microbiology, 112(2006), 275e279. Mortimore, S., & Smith, R. A. (1998). Standardized HACCP training: assurance for food authorities. Food Control, 9(2), 141e145. Motarjemi,Y.,&Käferstein,F.(1999).Foodsafety,hazardanalysisandcriticalcontrolpoint and increase in foodborne diseases: a paradox? Food Control, 10(4e5), 325e333. Nel, S., Lues, J. F. R., Buys, E. M., & Venter, P. (2004). The personal and general hygiene practices in the deboning room of a high throughput red meat abattoir. Food Control, 15(7), 571e578. Newcombe, R. G. (1998). Two-sided confidence intervals for the single proportion: comparison of seven methods. Statistics in Medicine, 17, 857e872. Nieto-Montenegro, S. N., Brown, J. L., & LaBorde, L. F. (2008). Development and assessment of pilot food safety educational materials and training strategies for Hispanic workers in the mushroom industry using the Health Action Model. Food Control, 19, 616e633. Notermans, S., Zwietering, M. H., & Mead, G. C. (1994). The HACCP concept: identifications of potentially hazardous microorganisms. FoodMicrobiology,11, 203e214. Pratten, J. D., & Curtis, S. (2002). Attitudes towards training in UK licensed retail: an exploratory case study. Hospitality Management, 21, 393e403. Regulation (EC) No 852/2004 of the European Parliament and of the Council of 29 of April 2004, on the hygiene of foodstuffs. Official Journal of European Commission. 01.02.2002 L31, pp. 1e24. Rennie, M. D. (1994). Evaluation of food hygiene education. British Food Journal, 96 (11), 20e25. Rennie, M. D. (1995). Health education models and food hygiene education. Journal of the Royal Society of Health, 115(2), 75e79. Rhodes, L. B. (1988). In M. E. Rhodes (Ed.), Competency-based adult learning in food safety programs in food protection technology II. Chelsea, MI: Lewis Publishers. Roberts, D. (1990). Foodborne illness, sources of infection: food. Lancet, 336, 859e861. Rusin, P., Maxwell, S., & Gerba, C. (2002). Comparative surface to-hand and fingertip-to-mouth transfer efficiency of gram-positive bacteria, gram-negative bacteria, phage. Journal of Applied Bacteriology, 93, 585e592. Scott, E., & Bloomfield, S. F. (1990). The survival and transfer of microbialcontamination via cloths, hands and utensils. Journal of Applied Bacteriology, 68, 271e278. Seaman, P. (2010). Food hygiene training: introducing the food hygiene training model. Food Control, 21, 381e387. Seaman, P., & Eves, A. (2006). The management of food safetydthe role of food hygiene training in the UK service sector. International Journal of Hospitality Management, 25, 278e296. Shojaei, H., Shooshtaripoor, J., & Amiri, M. (2006). Efficacy of simple hand-washing in reduction of microbial hand contamination of Iranian food handlers. Food Research International, 39, 525e529. Smith, R. (1994). Food hygiene training: the chance to create a coherent training policy. British Food Journal, 96(7), 41e45. Sneed, J., Strohbehn, C., Gilmore, S. A., & Mendonca, A. (2004). Microbiological evaluation of foodservice contact surfaces in Iowa assisted-living facilities. Journal of the American Dietetic Association, 104(11), 1722e1724. Sun, Y. M., & Ockerman, H. (2005). A review of the needs and current applications of HACCP system in foodservice areas. Food Control, 16(4), 325e332. Toh, P. S., & Birchenough, A. (2000). Food safety knowledge and attitudes: culture and environment impact on hawkers in Malaysia. Knowledge and attitudes are key attributes of concern in hawker food handling practices and outbreaks of food poisoning and their prevention. Food Control, 11, 447e452. Van Zyl, A. P. (1998). Red meat manual for veterinary public health. Pretoria: Directorate Veterinary Public Health. Walker, E., Pritchard, C., & Forsythe, S. (2003). Food handlers’hygiene knowledge in small food businesses. Food Control, 14(5), 339e343. Williams, A. P., Smith, R. A., Gaze, R., Mortimore, S. E., Motarjemi, Y., & Wallace, C. A. (2003). An international future for standards of HACCP training. Food Control, 14, 111e121. Wilson, E. B. (1927). Probable inference, the law of succession, and statistical inference. Journal of the American Statistical Association, 22, 209e212. World Health Organization. (2000). Foodborne disease: A focus for health education. Geneva: World Health Organization. World Health Organization. (2003). Eighth report 1999e2000 of WHO surveillance programme for control of food borne infections and intoxications in Europe. Available from. http://www.bfr.bund.de/internet/8threport/8threp_fr.htm. Worsfold, D. (2001). Food safety behaviour in butchers’shops. Nutrition & Food Science, 31(1), 13e18. E. Gomes-Neves et al. / Food Control 22 (2011) 501e507 507 SECTION IV – General Discussion 61 SECTION IV GENERAL DISCUSSION SECTION IV – General Discussion 63 Salmonella has ascended, in the last decades, as the worldwide most important cause of foodborne outbreaks and one of the most significant bacterial agents causing human disease. In the EU, real efforts have been achieved in order to control this development. Particularly, in fowl populations, by the implementation of specific control measures, a consistent descendent trend was obtained. However, swine and pork persist as important reservoirs of MDR Salmonella. The work presented in this PhD thesis answer several issues regarding the occurrence of MDR Salmonella in swine and pork, the cross-contamination process in the abattoir and the meat handlers level of knowledge and practice, concerning their participation in the overall problem. We present answers through the results below. In this study one hundred healthy pigs presented to slaughter in eight abattoirs in the north of Portugal (districts of Braga and Porto) were sampled (ileocecal lymph nodes, carcasses and meat). In the respective deboning room, the hands of the meat handler responsible for the cut and deboning process of each sampled carcass were also sampled (Chapter 1, Section II). In each abattoir, it was sampled one pig from every different batch presented to slaughter. It was possible to identify 64 holdings from 11 districts from the continental territory of Portugal. • What is the occurrence of Salmonella in Portuguese slaughter swine, carcasses, meat and meat handlers? • Is it possible to track Salmonella along different sampled material unveiling routes of cross-contamination? Salmonella was isolated in all abattoirs from both lymph nodes and carcasses, except for one abattoir where only one pig was sampled (Section III, Chapter 1). It was possible to observe a high Salmonella prevalence in slaughter swine (26%) and carcasses (16%), in accordance with previous Portuguese data (European Food Safety Authority, 2008; Vieira-Pinto et al., 2005). In the deboning rooms, Salmonella was isolated in 14% of meat samples and 9% of meat handlers’ hands. Nine different Salmonella enterica serotypes were detected in the 60 positive samples: S. Typhimurium (53.3%; n=32) and the monophasic variant S. 4,[5],12:i:- (5%; n=3), S. Derby, (18.3%; n=11), S. Rissen (6.6%; n=4), S. Mbandaka (5%; n=3), S. London (5%; n=3), S. Give (3.3%; n=2), S. Enteritidis (1.6%; n=1) and S. Sandiego (1.6%; n=1). In order to track Salmonella Salmonella spp. in swine – The abattoir as a link in the food chain 70 (Antunes et al., 2006), which suggest the dissemination of these genes, by horizontal spread, between food-producing animals, industrial facilities, namely abattoirs and meat plants, and eventually consumers, with meat handler’s participation. Eleven isolates of S. Derby, genetically related (type D1), were identified in swine from 4 abattoirs, and were associated with SSuT resistance phenotype, through the presence of aadA2, sul1, tet(A) encoding genes, all presented a class 1 integron (aadA2), also previously described in human and food products in Portugal (Antunes et al., 2006). S. London (type L1), recently identified in Portuguese swine (European Food Safety Authority, 2009, 2011a), presented the resistance phenotype ANSSuT and the genotype blatem-1, strA-strB, sul2 and tet(A), and was identified in both carcass and meat handler in two abattoirs. Clones of S. Mbandaka (types M1 and M2) presented resistance profile strA-strB, spread among three abattoirs. PFGE types T1, T3, T4, T6, T7, T8, D1, R1, and L1, which include 80% (n=48) of the isolates are mostly MDR (Section III, Capter2, Figure1). This study demonstrated clonal dissemination in different abattoirs and in diverse sampled materials, including meat handler’s hands, suggesting the transference of strains between pigs, abattoir environment and humans and unveiling an important public health problem. In this process, abattoir operators assume a double risk position: they are agents of cross-contamination, but they also can get the infection and transport it into the community. • What is the level of general knowledge and practice in meat handlers from slaughter houses and meat plants? To our knowledge, this study performed the first professional training survey in Portuguese meat handlers, through a self-administered questionnaire assessing “knowledge” and “practice” (Section III, Chapter 3). Answers were obtained from all the meat plants contacted, but 10% of the employees have not returned the questionnaire. In the present study, we have verified a low educational level of meat handlers, the average formal education years being 6.5 (in Portugal the mandatory formal education takes 12 years) in a group with a mean age of 35 (varying widely between 16-58). An important proportion of the meat handlers (72.7%) had professional training in two diverse areas: Good Practice in Food Industry (12.03%) and Work Safety and Hygiene (22.8%); 37.9% of the respondents have had training in both areas. Nevertheless, 24.5% of the respondents have never had training. Meat handlers with professional training in Good Practice in Food Industry (GPFI) and in both areas (BT) have had the highest proportions of correct answers in Knowledge (66.92 ± 16.36 and 67.26 ± 21.05, respectively) and Practice questions (70.53 ± 17.47 and 68.67 ± 22.58). The proportion of correct answers SECTION IV – General Discussion 71 in the MH groups who have had GPFI or BT training is significantly higher than the others from a statistical point of view, but an additional analysis of the content of the questions themselves (qualitative results) have also shown that WSH training is not pertinent to Food Hygiene and Food Safety knowledge and practice (in spite of being pertinent in terms of occupational safety and health). Concerning HACCP, which is a current and pertinent requirement of the EU Food Law, there was still a high proportion of MH (even with professional training, the WSH group) who was unaware of the concept. To the question “What is HACCP,” only half of BT had answered correctly and this group has also had the highest proportion of incorrect answers, conflicting to what should be predictable. It appears to be very problematic to implement an HACCP based system in this industry, when a high proportion of employees is not acquainted with this reality and does not participate in pre-requisite programs. The results of this study point to the necessity to develop training, particularly in Good Practice in Food Industry, thus allowing meat handlers to improve their Knowledge and Practice. The implementation of evaluation criteria for the effectiveness of professional training is also critical as there is no evidence that the worker practices improve when training programs provide only information. What we have observed in the present study is that the pressure to accomplish the law leads employers to get specialized training for their employees. However, the success of training relies on the choice of the program, considering the relevance of the course to work activities, and providing food hygiene training in a language and at a level that allows the food handler to understand the content. Regarding the level of formal education observed within the respondent group, this limitation should be considered. There is also a need to develop training methods that proved to change behavior as well as imparting knowledge, considering that only 50.3% of MH with professional training believes that training provides useful information to their work. Furthermore, Food Industry operators cannot rely on the fact that training has ever taken place. They must assume that all employees will need detailed, repeated refreshment training in the area of food hygiene and safety, in spite of the previous work experience. In the present study, the meat handlers demonstrated an average of 12.6 years of experience in the activity. Nevertheless, the respondents have had poor results on the HACCP, microbiological hazards, temperature control, and personal hygiene and cross contamination subjects. Regarding the importance of the participation of meat handlers in the Food Chain and considering that 64.9% admitted their interest in future training the improvement opportunity should not be neglected. SECTION V – Conclusions and Perspectives 73 SECTION V CONCLUSIONS AND PERSPECTIVES SECTION V – Conclusions and Perspectives 75 This work describes evidence founded on original studies of the occurrence and characterization of MDR Salmonella isolates in slaughter swine products and meat handlers, and on the first Portuguese meat handlers’ knowledge and practice survey. Based on these primary results, it seems appropriated, as final conclusions and perspectives, to refer: Our data suggest that swine and pork meat are a significant source of MDR Salmonella and that abattoir processes could endorse its contamination. While swine could harbor Salmonella previously to slaughter, the abattoir environment can contribute to further cross-contamination along the slaughter line, including contact with meat handlers. In order to improve standards in the post-harvest pork meat chain, measures have to be taken at these stages. Reducing the prevalence of Salmonella positive pigs at the primary production phase can significantly decrease one of the main sources of contamination at the abattoir. However, strong evidence is clear: hygiene of slaughter operations, and meat handlers’ practice should also be improved. Guidelines and procedures to reduce Salmonella are an essential requisite in abattoirs and the warranty of its accomplishment is critical. Moreover, they should highlight the requirement for safe food-handling practices in abattoirs and meat plants to reduce the level of Salmonella occurrence in carcasses and pork, emphasizing the role of the meat handlers’ effective training and the control of the implementation of an improved practice. Meat handlers’ training, as it has been generally developed in the abattoir and meat plants' context, it is not being an efficient tool to acquire a robust knowledge and to adopt a safe practice. As previously referred, in this activity, characterized by hard physical work and a traditionally low educational level of the workers, professional training should be adapted, with a strong connection knowledge-practice, considering motivational factors and beliefs. Behavior changes should be evaluated according to those conditions, encouraging the learning process and rewarding practical improvements, namely towards a safe handling goal. Additionally, GMP and current HACCP implementation remain crucial to reduce cross-contamination and to maintain the level of Salmonella burden as low as possible in abattoir environment and pork meat, protecting public health. Cooperation between Veterinary Authorities and abattoir and meat plants’ Operators, is required in order to achieve effective advances in meat safety. The identification of MDR Salmonella clones in swine, pork and meat handlers, as well as the appearance of emergent international clones, namely S. Typhimurium DT104, the monophasic variant S. 4,[5],12:i:- and S. Rissen with wide resistance patterns, is of Salmonella spp. in swine – The abattoir as a link in the food chain 76 great concern. This fact recognizes an insufficiently studied transmission path, relevant in public health, and requires an intervention. Furthermore, the circulation of animals and food products all over the EU multiplies the possibilities of acquisition of new strains and intensifies this MDR threat. The surveillance of antimicrobial resistance to follow the emergence and spread of MDR Salmonella from these sources seems critical. Particularly, S. 4,[5],12:i:-, here primarily reported with a new resistance phenotype, and assuming an increasing relevance worldwide, has been scarcely characterized and requires a close surveillance at a national level. S. London, recently identified in an EU base line survey in Portuguese swine, to our knowledge, firstly reported in this work with a wide MDR pattern, requires to be followed. Our results, revealing high frequency of MDR phenotypes to veterinary most used antibiotics, support the need of a revision of preventive measures, infection-control strategies and interventions in primary production (e.g. biosecurity, vaccination, good surveillance, rapid detection and treatment). The development of cooperation programs involving Veterinary Authorities and veterinarians in swine herds is essential. These programs should include monitoring antimicrobial susceptibility and MDR among Salmonella spp. isolated from environment and clinically ill animals. This assessment should comprise molecular characterization with structural and functional data, which are essential for understanding the emergence of new resistance mechanisms and virulence, and to control its spread into the food chain and the community. SECTION VI – References 77 SECTION VI REFERENCES SECTION VI – References 79 Acheson, D., & Hohmann, E. L. (2001). Nontyphoidal salmonellosis. Clinical Infectious Diseases, 32(2), 263-269. Alban, L., Stege, H., & Dahl, J. (2002). The new classification system for slaughter-pig herds in the Danish Salmonella surveillance-and-control program. Preventive Veterinary Medicine, 53(1-2), 133-146. Alcaine, S. D., Warnick, L. D., & Wiedmann, M. (2007). Antimicrobial resistance in nontyphoidal Salmonella. Journal of Food Protection, 174, 70(3), 780-790. Allwood, P. B., Jenkins, T., Paulus, C., Johnson, L., & Hedberg, C. W. (2004). Hand washing compliance among retail food establishment workers in Minnesota. J Food Prot, 67(12), 2825-2828. Amyes, S. G. B. (2010). Antibacterial Chemotherapy: Theory, Problems, and Practice: Oxford Univ Pr. Anderson, E., Ward, L. R., Saxe, M. J., & De Sa, J. (1977). Bacteriophage-typing designations of Salmonella typhimurium. J Hyg (Lond), 78(2), 297-300. Anjum, M. F., Choudhary, S., Morrison, V., Snow, L. C., Mafura, M., Slickers, P., Woodward, M. J. (2011). Identifying antimicrobial resistance genes of human clinical relevance within Salmonella isolated from food animals in Great Britain. J. Antimicrob. Chemother., 66(3), 550-559. doi: 10.1093/jac/dkq498 Antunes, P., Machado, J., & Peixe, L. (2006). Characterization of antimicrobial resistance and class 1 and 2 integrons in Salmonella enterica isolates from different sources in Portugal. Journal of Antimicrobial Chemotherapy, 58(2), 297-304. Antunes, P., Mourão, J., Pestana, N., & Peixe, L. (2011). Leakage of emerging clinically relevant multidrug-resistant Salmonella clones from pig farms. [Research Support, Non-U.S. Gov’t]. J Antimicrob Chemother, 66(9), 2028-2032. doi: 10.1093/jac/ dkr228 Arguello, H., Carvajal, A., Collazos, J. A., García-Feliz, C., & Rubio, P. (2011). Prevalence and serovars of Salmonella enterica on pig carcasses, slaughtered pigs and the environment of four Spanish slaughterhouses. Food Research International. Arnold, S., Gassner, B., Giger, T., & Zwahlen, R. (2004). Banning antimicrobial growth promoters in feedstuffs does not result in increased therapeutic use of antibiotics in medicated feed in pig farming. Pharmacoepidemiology and Drug Safety, 13(5), 323- 331. doi: 10.1002/pds.874 Salmonella spp. in swine – The abattoir as a link in the food chain 86 Fluit, A. C. (2005). Towards more virulent and antibiotic resistant Salmonella? FEMS Immunology & Medical Microbiology, 43(1), 1-11. Fosse, J., Seegers, H., & Magras, C. (2009). Prevalence and Risk Factors for Bacterial Food-Borne Zoonotic Hazards in Slaughter Pigs: A Review. Zoonoses and Public Health, 56(8), 429-454. Foster, N., Lovell, M., Marston, K., Hulme, S., Frost, A., Bland, P., & Barrow, P. (2003). Rapid protection of gnotobiotic pigs against experimental salmonellosis following induction of polymorphonuclear leukocytes by avirulent Salmonella enterica. Infect Immun, 71(4), 2182-2191. Foster, N., Hulme, S., Lovell, M., Reed, K., & Barrow, P. (2005). Stimulation of gp91 phagocytic oxidase and reactive oxygen species in neutrophils by an avirulent Salmonella enterica serovar infantis strain protects gnotobiotic piglets from lethal challenge with serovar Typhimurium strain F98 without inducing intestinal pathology. Infect Immun, 73(8), 4539-4547. Foti, M., Daidone, A., Aleo, A., Pizzimenti, A., Giacopello, C., & Mammina, C. (2009). Salmonella bongori 48: z35:–in Migratory Birds, Italy. Emerging Infectious Diseases, 15(3), 502. Franz, E., & Van Bruggen, A. H. C. (2008). Ecology of E. coli O157: H7 and Salmonella enterica in the primary vegetable production chain. Critical Reviews in Microbiology, 34(3-4), 143-161. Fraser, D. (2006). Animal welfare assurance programs in food production: a framework for assessing the options. Animal Welfare, 15(2), 93. Fry, A., Braden, C., Griffin, P., & Hughes, J. (2005). Foodborne disease. In Mandell G. L., Bennett J. E. & Dolin R. (Eds.) , Principles and practice of infectious diseases. (6th ed., pp. 1286-1297). New York: Elsevier, Churchill Livingstone. Fullerton, K. (2008). Monitoring the incidence and causes of diseases potentially transmitted by food in Australia: annual report of the OzFoodNet Network, 2007. Commun Dis Intell, 32(4), 400-424. Galán, J. E. (2001). Salmonella interactions with host cells: type III secretion at work. Annual Review of Cell and Developmental Biology, 17(1), 53-86. García-Feliz, C., Collazos, J. A., Carvajal, A., Herrera, S., Echeita, M. A., & Rubio, P. (2008). Antimicrobial Resistance of Salmonella enterica Isolates from Apparently Healthy and Clinically Ill Finishing Pigs in Spain. Zoonoses and Public Health, 55(4), 195-205. doi: 10.1111/j.1863-2378.2008.01110.x SECTION VI – References 87 García-Feliz, C., Carvajal, A., Collazos, J. Á., & Rubio, P. (2009). Herd-level risk factors for faecal shedding of Salmonella enterica in Spanish fattening pigs. Preventive Veterinary Medicine, 91(2-4), 130-136. Gebreyes, W. (2008). Salmonella in Swine. Food Safety Research and Response Network Journal, IV(1), 1-7. Gebreyes, W. A., Thakur, S., Davies, P. R., Funk, J. A., & Altier, C. (2004). Trends in antimicrobial resistance, phage types and integrons among Salmonella serotypes from pigs, 1997-2000. J Antimicrob Chemother, 53(6), 997-1003. doi: 10.1093/jac/ dkh247 Giaccone, V., Catellani, P., & Alberghini, L. (2012). Food as Cause of Human Salmonellosis. In B. S. M. Mahmoud (Ed.), Salmonella - A Dangerous Foodborne Pathogen (pp. 47-72): InTech, January, 2012. Giannella, R., Broitman, S., & Zamcheck, N. (1972). Gastric acid barrier to ingested microorganisms in man: studies in vivo and in vitro. Gut, 13(4), 251. Giovannacci, I., Queguiner, S., Ragimbeau, C., Salvat, G., Vendeuvre, J., Carlier, V., & Ermel, G. (2001). Tracing of Salmonella spp. in two pork slaughter and cutting plants using serotyping and macrorestriction genotyping. Journal of Applied Microbiology, 90(1), 131-147. Gopinath, S., Carden, S., & Monack, D. (2012). Shedding light on Salmonella carriers. Trends in microbiology, 20(7), 320-327. doi: 10.1016/j.tim.2012.04.004 Gould, I. (1999). A review of the role of antibiotic policies in the control of antibiotic resistance. Journal of Antimicrobial Chemotherapy, 43(4), 459-465. Graziani, C., Busani, L., Dionisi, A. M., Lucarelli, C., Owczarek, S., Ricci, A., Luzzi, I. (2008). Antimicrobial resistance in Salmonella enterica serovar Typhimurium from human and animal sources in Italy. Veterinary Microbiology, 128(3-4), 414-418. doi: 10.1016/j.vetmic.2007.10.017 Greenberg, B., Kowalski, J. A., & Klowden, M. J. (1970). Factors affecting the transmission of Salmonella by flies: natural resistance to colonization and bacterial interference. Infect Immun, 2(6), 800-809. Griffith, R. W., Schwartz, K. J. & Meyerholz, D. K. (2006). Salmonella. Diseases of Swine (9th ed.): Blackwell Publishing Ltd. Grimont, P. A. D., & Weill, F. X. (2007). Antigenic formulae of the Salmonella serovars. WHO Collaborating Centre for Reference and Research on Salmonella Institut Pasteur, Paris, France. Salmonella spp. in swine – The abattoir as a link in the food chain 88 Guardabassi, L., & Kruse, H. (2009). Principles of Prudent and Rational Use of Antimicrobials in Animals. Guide to Antimicrobial Use in Animals (pp. 1-12): Blackwell Publishing, Ltd. Gudding, R. (1975). The persistence of Salmonella Typhimurium in various types of manure with and without admixture of silage effluent. Acta Veterinaria Scandinavica, 16(1), 115. Haesebrouck, F., Pasmans, F., Chiers, K., Maes, D., Ducatelle, R., & Decostere, A. (2004). Efficacy of vaccines against bacterial diseases in swine: what can we expect? Veterinary Microbiology, 100(3-4), 255-268. doi: 10.1016/j.vetmic.2004.03.002 Hald, T., Lo Fo Wong, D. M. A., & Aarestrup, F. M. (2007). The attribution of human infections with antimicrobial resistant Salmonella bacteria in Denmark to sources of animal origin. Foodborne Pathogens and Disease, 4(3), 313-326. Hall, R. M., & Collis, C. M. (1998). Antibiotic resistance in gram-negative bacteria: the role of gene cassettes and integrons. Drug resistance UPDATES, 1(2), 109-119. Hamilton, D., Smith, P., & Pointon, A. (2007). National Salmonella and E. coli Monitoring (ESAM) data from Australian pig carcases from 2000 to 2006. Hancock, E. (2012). Importance of adaptive and stepwise changes in the rise and spread of antimicrobial resistance. Antimicrobial Resistance in the Environment, 43. Hanssen, E., Swanenburg, M., & Maassen, C. (2007). The Dutch Salmonella monitoring programme for pigs and some recommendations for control plans in the future: Wageningen University and Research Center (http://library.ur.nl/oai) (Netherlands) ER (acessed May 13, 2012). Haraga, A., Ohlson, M. B., & Miller, S. I. (2008). Salmonellae interplay with host cells. Nature Reviews Microbiology, 6(1), 53-66. Hauser, E., Tietze, E., Helmuth, R., Junker, E., Blank, K., Prager, R., Malorny, B. (2010). Pork contaminated with Salmonella enterica serovar 4,[5],12:i:-, an emerging health risk for humans. Appl Environ Microbiol, 76(14), 4601-4610. doi: 10.1128/aem.02991-09 Helms, M., Ethelberg, S., & Mølbak, K. (2005). International Salmonella Typhimurium DT104 infections, 1992-2001. Emerging Infectious Diseases, 11(6), 859. Hendriksen, R. S., Bangtrakulnonth, A., Pulsrikarn, C., Pornreongwong, S., Hasman, H., Song, S. W., & Aarestrup, F. M. (2008). Antimicrobial resistance and molecular epidemiology of Salmonella Rissen from animals, food products, and patients in Thailand and Denmark. Foodborne Pathogens and Disease, 5(5), 605-619. SECTION VI – References 89 Hendriksen, R. S., Vieira, A. R., Karlsmose, S., Lo Fo Wong, D. M. A., Jensen, A. B., Wegener, H. C., & Aarestrup, F. M. (2011). Global monitoring of Salmonella serovar distribution from the World Health Organization Global Foodborne Infections Network country data bank: results of quality assured laboratories from 2001 to 2007. Foodborne Pathogens and Disease, 8(8), 887-900. Hensel, M. (2000). Salmonella pathogenicity island 2. Mol Microbiol, 36(5), 1015-1023. Hidalgo-Vila, J., Díaz-Paniagua, C., de Frutos-Escobar, C., Jiménez-Martínez, C., & Pérez-Santigosa, N. (2007). Salmonella in free living terrestrial and aquatic turtles. Veterinary Microbiology, 119(2), 311-315. Hill, A., England, T., Snary, E., Cook, A., Kelly, L., Evans, S., & Wooldridge, M. (2003). A ‘farm-to-consumption’ risk assessment for Salmonella Typhimurium in pigs. Department of Risk Research, Veterinary Laboratories Agency: Department of Risk Research, Veterinary Laboratories Agency. Weybridge. Höjgård, S., & Vågsholm, I. (2010). Antimicrobial sensitivity as a natural resource and global public good-Resistance as an externality: Agrifood working paper available at http://www. agrifood. se/publications. aspx (accessed May 22, 2012). Holley, R. A., & Guan, T. Y. (2003). Pathogen survival in swine manure environments and transmission of human enteric illness - a review. Journal of Environmental Quality, 32(2), 383-392. Hoogstral, H. (1986). Theobald Smith: his scientific work and impact. Bulletin of the ESA, 32(1), 22-35. Hopkins, K. L., Kirchner, M., Guerra, B., Granier, S. A., Lucarelli, C., Porrero, M. C., Mevius, D. J. (2010). Multiresistant Salmonella enterica serovar 4,[5],12:i:- in Europe: a new pandemic strain? Euro Surveill, 15(22), 19580. doi: 19580 [pii] Hsu, H. (1989). Pathogenesis and immunity in murine salmonellosis. Microbiology and Molecular Biology Reviews, 53(4), 390. Hsu, R. B., Tsay, Y. G., Chen, R. J., & Chu, S. H. (2003). Risk factors for primary bacteremia and endovascular infection in patients without acquired immunodeficiency syndrome who have nontyphoid salmonellosis. Clinical Infectious Diseases, 36(7), 829. Huang, T. M., Lin, T., & Wu, C. (2009). Serovar distribution and antimicrobial susceptibility of swine Salmonella isolates from clinically ill pigs in diagnostic submissions from Indiana in the United States. Lett Appl Microbiol, 48(3), 331-336. Hur, J., Jawale, C., & Lee, J. H. (2012). Antimicrobial resistance of Salmonella isolated from food animals: A review. Food Research International, 45(2), 819-830. doi: 10.1016/j.foodres.2011.05.014 Salmonella spp. in swine – The abattoir as a link in the food chain 90 Hurd, H. S., McKean, J. D., Wesley, I. V., & Karriker, L. A. (2001). The effect of lairage on Salmonella isolation from market swine. Journal of Food Protection, 174;, 64(7), 939-944. Isaacson, R., Firkins, L., Weigel, R., Zuckermann, F., & DiPietro, J. (1999). Effect of transportation and feed withdrawal on shedding of Salmonella Typhimurium among experimentally infected pigs. American Journal of Veterinary Research, 60(9), 1155. Islam, M., Morgan, J., Doyle, M. P., Phatak, S. C., Millner, P., & Jiang, X. (2004). Persistence of Salmonella enterica serovar Typhimurium on lettuce and parsley and in soils on which they were grown in fields treated with contaminated manure composts or irrigation water. Foodborne Pathogens & Disease, 1(1), 27-35. Jansen, A., Frank, C., & Stark, K. (2007). Pork and pork products as a source for human salmonellosis in Germany. Berliner und Münchener Tierärztliche Wochenschrift, 120(7-8), 340. Jansen, W., Van der Bruggen, J., Verhoef, J., & Fluit, A. (2006). Bacterial resistance: A sensitive issue: Complexity of the challenge and containment strategy in Europe. Drug resistance Updates, 9(3), 123-133. Johnsen, P. J., Townsend, J. P., Bøhn, T., Simonsen, G. S., Sundsfjord, A., & Nielsen, K. M. (2009). Factors affecting the reversal of antimicrobial-drug resistance. The Lancet Infectious Diseases, 9(6), 357-364. Kich, J. D., Schwarz, P., Silva, L. E., Coldebella, A., Piffer, I. A., & Vizzoto, R. (2007). Development and application of an enzyme-linked immunosorbent assay to detect antibodies against prevalent Salmonella serovars in swine in southern Brazil. Journal of Veterinary Diagnostic Investigation, 19(5), 510. Kich, J. D., Coldebella, A., Mores, N., Nogueira, M. G., Cardoso, M., Fratamico, P. M., Luchansky, J. B. (2011). Prevalence, distribution, and molecular characterization of Salmonella recovered from swine finishing herds and a slaughter facility in Santa Catarina, Brazil. [Research Support, Non-U.S. Gov’t Research Support, U.S. Gov’t, Non-P.H.S.]. Int J Food Microbiol, 151(3), 307-313. doi: 10.1016/j.ijfoodmicro.2011.0 9.024 King, N., Lake, R., & Campbell, D. (2011). Source Attribution of Nontyphoid Salmonellosis in New Zealand Using Outbreak Surveillance Data. Journal of Food Protection, 174; 74(3), 438-445. doi: 10.4315/0362-028x.jfp-10-323 Klerks, M. M., Franz, E., van Gent-Pelzer, M., Zijlstra, C., & Van Bruggen, A. H. C. (2007). Differential interaction of Salmonella enterica serovars with lettuce cultivars and plant-microbe factors influencing the colonization efficiency. The ISME Journal, 1(7), 620-631. SECTION VI – References 91 Kühnel, K., & Blaha, T. (2004). Investigations on targeted intervention measures for minimizing Salmonella cross-contamination during slaughter. Paper presented at the Proceedings of the 18th IPVS Congress, Hamburg, Germany. Laine, T., Yliaho, M., Myllys, V., Pohjanvirta, T., Fossi, M., & Anttila, M. (2004). The effect of antimicrobial growth promoter withdrawal on the health of weaned pigs in Finland. Preventive Veterinary Medicine, 66(1-4), 163-174. doi: 10.1016/ j.prevetmed.2004.09.001 Letellier, A., Messier, S., Paré, J., Ménard, J., & Quessy, S. (1999). Distribution of Salmonella in swine herds in Québec. Veterinary Microbiology, 67(4), 299-306. doi: 10.1016/s0378-1135(99)00049-8 Levin, B. R. (2001). Minimizing potential resistance: a population dynamics view. Clinical Infectious Diseases, 33(Supplement 3), S161-S169. Levy, S. B. (1984). Playing antibiotic pool: time to tally the score. New England Journal of Medicine, 311(10), 663-665. Levy, S. B. (2001). Antibiotic resistance: consequences of inaction. Clinical Infectious Diseases, 33(Supplement 3), S124-S129. Liang-Takasaki, C., Saxen, H., Makela, P., & Leive, L. (1983). Complement activation by polysaccharide of lipopolysaccharide: an important virulence determinant of salmonellae. Infect Immun, 41(2), 563. Liebana, E., Garcia-Migura, L., Breslin, M. F., Davies, R. H., & Woodward, M. J. (2001). Diversity of strains of Salmonella enterica serotype Enteritidis from English poultry farms assessed by multiple genetic fingerprinting. Journal of Clinical Microbiology, 39(1), 154-161. Liebana, E., Garcia-Migura, L., Clouting, C., Cassar, C., Clifton-Hadley, F., Lindsay, E.,... Davies, R. (2002). Investigation of the genetic diversity among isolates of Salmonella enterica serovar Dublin from animals and humans from England, Wales and Ireland. Journal of Applied Microbiology, 93(5), 732-744. Lo Fo Wong, D., Dahl, J., Stege, H., Van Der Wolf, P., Leontides, L., Von Altrock, A., & Thorberg, B. (2004). Herd-level risk factors for subclinical Salmonella infection in European finishing-pig herds. Preventive Veterinary Medicine, 62(4), 253-266. Lo Fo Wong, D. M. A., Hald, T., van der Wolf, P. J., & Swanenburg, M. (2002). Epidemiology and control measures for Salmonella in pigs and pork. Livestock Production Science, 76(3), 215-222. doi: 10.1016/s0301-6226(02)00121-5 Salmonella spp. in swine – The abattoir as a link in the food chain 92 Lo Fo Wong, D. M. A., Dahl, J., van der Wolf, P. J., Wingstrand, A., Leontides, L., & von Altrock, A. (2003). Recovery of Salmonella enterica from seropositive finishing pig herds. Vet Microbiol, 97(3-4), 201-214. doi: 10.1016/j.vetmic.2003.09.012 Lucarelli, C., Dionisi, A. M., Torpdahl, M., Villa, L., Graziani, C., Hopkins, K., Luzzi, I. (2010). Evidence for a second genomic island conferring multidrug resistance in a clonal group of strains of Salmonella enterica serovar Typhimurium and its monophasic variant circulating in Italy, Denmark, and the United Kingdom. J Clin Microbiol, 48(6), 2103-2109. doi: JCM.01371-09 [pii] 10.1128/JCM.01371-09 Lucarelli, C., Dionisi, A. M., Filetici, E., Owczarek, S., Luzzi, I., & Villa, L. (2012). Nucleotide sequence of the chromosomal region conferring multidrug resistance (R- type ASSuT) in Salmonella Typhimurium and monophasic Salmonella Typhimurium strains. J Antimicrob Chemother, 67(1), 111-114. doi: 10.1093/jac/dkr391 Lues, J. F. R., & Van Tonder, I. (2007). The occurrence of indicator bacteria on hands and aprons of food handlers in the delicatessen sections of a retail group. Food Control, 18(4), 326-332. doi: 10.1016/j.foodcont.2005.10.010 Maes, D., Gibson, K., Trigo, E., Saszak, A., Grass, J., Carlson, A., & Blaha, T. (2001). Evaluation of cross-protection afforded by a Salmonella Choleraesuis vaccine against Salmonella infections in pigs under field conditions. Berliner und Münchener Tierärztliche Wochenschrift, 114(9-10), 339. Manageiro, V. (2011). Dynamics of β-lactamases in Gram-negative bacteria. PhD Thesis, Faculdade de Ciências - Universidade de Lisboa, Lisboa. Mandell, G. L., Douglas Jr, R. G., & Bennett, J. E. (1979). Principles and Practice of Infectious Diseases. Volumes 1 and 2: John Wiley & Sons. Mann, J., Smith, L., & Brashears, M. (2004). Validation of time and temperature values as critical limits for Salmonella and background flora growth during the production of fresh ground and boneless pork products. Journal of Food Protection, 174; 67(7), 1389-1393. Mannion, C., Leonard, F., Lynch, P., & Egan, J. (2007). Efficacy of cleaning and disinfection on pig farms in Ireland. Veterinary Record, 161(11), 371-375. Marg, H., Scholz, H., Arnold, T., Rösler, U., & Hensel, A. (2001). Influence of longtime transportation stress on re-activation of Salmonella typhimurium DT104 in experimentally infected pigs. Berliner und Münchener Tierärztliche Wochenschrift, 114(9-10), 385. Mastroeni, P., Chabalgoity, J. A., Dunstan, S. J., Maskell, D. J., & Dougan, G. (2001). Salmonella: immune responses and vaccines. [Review]. Vet J, 161(2), 132-164. doi: 10.1053/tvjl.2000.0502 SECTION VI – References 93 Mastroeni, P., & Ménager, N. (2003). Development of acquired immunity to Salmonella. Journal of Medical Microbiology, 52(6), 453-459. Mastroeni, P., & Maskell, D. (2006). Salmonella infections: clinical, immunological, and molecular aspects. Cambridge, UK ; New York: Cambridge University Press. McDonnell, G., & Russell, A. D. (1999). Antiseptics and disinfectants: activity, action, and resistance. Clin Microbiol Rev, 12(1), 147-179. McEwen, S. A. (2009). Antibiotic Resistance – The Global Perspective. Advances in Pork Production, 20, 183-190. Merle, R., Schneider, B., Franz, B., Portsch, U., May, T., Blaha, T., & Kreienbrock, L. (2006). The serological Salmonella monitoring in German pork production: the structure of the central database and preliminary results of a basic epidemiological report. United Kingdom, retrieved from http://www.svepm.org.uk (acessed March 10, 2012) Merle, R., Kösters, S., May, T., Portsch, U., Blaha, T., & Kreienbrock, L. (2011). Serological Salmonella monitoring in German pig herds: Results of the years 2003- 2008. Preventive Veterinary Medicine, 99(2-4), 229-233. Millemann, Y., Lesage, M. C., Chaslus-Dancla, E., & Lafont, J. P. (1995). Value of plasmid profiling, ribotyping, and detection of IS200 for tracing avian isolates of Salmonella typhimurium and S. enteritidis. Journal of Clinical Microbiology, 33(1), 173-179. Miller, G. Y., Liu, X., McNamara, P. E., & Barber, D. A. (2005). Influence of Salmonella in pigs preharvest and during pork processing on human health costs and risks from pork. Journal of Food Protection, 174; 68(9), 1788-1798. Miriagou, V., Carattoli, A., & Fanning, S. (2006). Antimicrobial resistance islands: resistance gene clusters in Salmonella chromosome and plasmids. Microbes and Infection, 8(7), 1923-1930. doi: DOI 10.1016/j.micinf.2005.12.027 Mitscherlich, E., & Marth, E. H. (1984). Microbial survival in the environment. Bacteria and rickettsiae important in human and animal health: Springer-Verlag; CAB direct.org. Mølbak, K., Baggesen, D. L., Aarestrup, F. M., Ebbesen, J. M., Engberg, J., Frydendahl, K., Wegener, H. C. (1999). An outbreak of multidrug-resistant, quinolone-resistant Salmonella enterica serotype Typhimurium DT104. New England Journal of Medicine, 341(19), 1420-1425. Mølbak, K., Olsen, J. E., & Wegener, H. C. (2006). Salmonella infections. In Hans P. Rieman & Dean O. Cliver (Ed.), Foodborne Infections and Intoxications. (3rd. ed., pp. 57-136): Elsevier. Salmonella spp. in swine – The abattoir as a link in the food chain 94 Molla, B., Sterman, A., Mathews, J., Artuso-Ponte, V., Abley, M., Farmer, W., Gebreyes, W. A. (2010). Salmonella enterica in commercial swine feed and subsequent isolation of phenotypically and genotypically related strains from fecal samples. Applied and Environmental Microbiology, 76(21), 7188-7193. Møretrø, T., Heir, E., Nesse, L. L., Vestby, L. K., & Langsrud, S. (2012). Control of Salmonella in food related environments by chemical disinfection. Food Research International, 45(2), 532-544. doi: 10.1016/j.foodres.2011.02.002 Mossel, D., Morris, G., Struijk, C., Cowden, J., & Browning, L. (2003). Providing an Adequate Supply of Microbiologically Safe and Palatable Food and Drinking Water: Contribution of a European Vertically Integrated Approach to Educating Professionals and Consumers – Part 2. Food Protection Trends, 23(2). Nastasi, A., Mammina, C., & Salsa, L. (1999). Outbreak of Salmonella enteritis bongori 48: z35:-in Sicily. Euro surveillance: bulletin europeen sur les maladies transmissibles. European Communicable Disease Bulletin, 4(9), 97. Nel, S., Lues, J. F. R., Buys, E. M., & Venter, P. (2004). The personal and general hygiene practices in the deboning room of a high throughput red meat abattoir. Food Control, 15(7), 571-578. doi: 10.1016/j.foodcont.2003.09.004 Newell, D. G., Koopmans, M., Verhoef, L., Duizer, E., Aidara-Kane, A., Sprong, H., Scheutz, F. (2010). Food-borne diseases – The challenges of 20 years ago still persist while new ones continue to emerge. International Journal of Food Microbiology, 139, S3-S15. Nielsen, B., Alban, L., Stege, H., Sørensen, L., Møgelmose, V., Bagger, J., Baggesen, D. L. (2001). A new Salmonella surveillance and control programme in Danish pig herds and slaughterhouses. Berliner und Münchener Tierärztliche Wochenschrift, 114(9-10), 323. Nollet, N., Maes, D., De Zutter, L., Duchateau, L., Houf, K., Huysmans, K., Van Hoof, J. (2004). Risk factors for the herd-level bacteriologic prevalence of Salmonella in Belgian slaughter pigs. Preventive Veterinary Medicine, 65(1-2), 63-75. doi: 10.1016/ j.prevetmed.2004.06.009 Nollet, N., Maes, D., Duchateau, L., Hautekiet, V., Houf, K., Van Hoof, J., Geers, R. (2005). Discrepancies between the isolation of Salmonella from mesenteric lymph nodes and the results of serological screening in slaughter pigs. Vet Res, 36(4), 545-555. Nollet, N., Houf, K., Dewulf, J., Catry, B., De Zutter, L., De Kruif, A., & Maes, D. (2006). Variability in antimicrobial resistance among Salmonella enterica strains from fattening pigs and sows. Microbial Drug Resistance, 12(1), 74-81. SECTION VI – References 95 O’Connor, A., Denagamage, T., Sargeant, J., Rajić, A., & McKean, J. (2008). Feeding management practices and feed characteristics associated with Salmonella prevalence in live and slaughtered market-weight finisher swine: A systematic review and summation of evidence from 1950 to 2005. Preventive Veterinary Medicine, 87(3), 213-228. Ojha, S., & Kostrzynska, M. (2007). Approaches for reducing Salmonella in pork production. Journal of Food Protection, 174; 70(11), 2676-2694. Olsen, J. E., Skov, M. N., Angen, Ø., Threlfall, E. J., & Bisgaard, M. (1997). Genomic relationships between selected phage types of Salmonella enterica subsp. enterica serotype typhimurium defined by ribotyping, IS200 typing and PFGE. Microbiology, 143(4), 1471-1479. On, S., & Baggesen, D. L. (1997). Determination of clonal relationships of Salmonella typhimurium by numericalanalysis of macrorestriction profiles. Journal of Applied Microbiology, 83(6), 699-706. Österberg, J. (2010). Salmonella in pigs – Infection Dynamics of Different Serotypes. PhD thesis, Faculty of Veterinary Medicine and Animal Sciences. Department of Clinical Sciences. Uppsala – Swedish University of Agricultural Sciences. Padungtod, P., & Kaneene, J. B. (2006). Salmonella in food animals and humans in northern Thailand. International Journal of Food Microbiology, 108(3), 346-354. Painter, J. A., Molbak, K., Sonne-Hansen, J., Barrett, T., Wells, J. G., & Tauxe, R. V. (2004). Salmonella – based rodenticides and public health. Emerg Infect Dis, 10(6), 985-987. Parry, C. M., & Threlfall, E. (2008). Antimicrobial resistance in typhoidal and nontyphoidal salmonellae. Current Opinion in Infectious Diseases, 21(5), 531-538 510.1097/QCO. 1090b1013e32830f32453a. Phillips, I., Casewell, M., Cox, T., De Groot, B., Friis, C., Jones, R., Waddell, J. (2004). Does the use of antibiotics in food animals pose a risk to human health? A critical review of published data. Journal of Antimicrobial Chemotherapy, 53(1), 28-52. Phillips, I. (2007). Withdrawal of growth-promoting antibiotics in Europe and its effects in relation to human health. International Journal of Antimicrobial Agents, 30(2), 101- 107. doi: 10.1016/j.ijantimicag.2007.02.018 Popoff, M. Y., & Le Minor, L. (1997). Formules antigéniques des sérovars de Salmonella: Antigenic formulas of the Salmonella serovars: WHO collaborating centre for reference and research on Salmonella. Popoff, M. Y., Bockemuhl, J., & Gheesling, L. L. (2003). Supplement 2001 (no. 45) to the Kauffmann-White scheme. Research in Microbiology, 154(3), 173-174.